Mutant ROS Expression In Human Cancer

ABSTRACT

The invention provides the identification of the presence of mutant ROS protein in human cancer. In some embodiments, the mutant ROS are FIG-ROS fusion proteins comprising part of the FIG protein fused to the kinase domain of the ROS kinase. In some embodiments, the mutant ROS is the overexpression of wild-type ROS in cancerous tissues (or tissues suspected of being cancerous) where, in normal tissue of that same tissue type, ROS is not expressed or is expressed at lower levels. The mutant ROS proteins of the invention are anticipated to drive the proliferation and survival of a subgroup of human cancers, particularly in cancers of the liver (including bile duct), pancreas, kidney, and testes. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant ROS polypeptides (e.g., a FIG-ROS(S) fusion polypeptide), probes for detecting it, isolated mutant polypeptides, recombinant polypeptides, and reagents for detecting the fusion and truncated polypeptides. The identification of the mutant ROS polypeptides enables new methods for determining the presence of these mutant ROS polypeptides in a biological sample, methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer characterized by the mutant polynucleotides or polypeptides, which are also provided by the invention.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalpatent application U.S. Ser. No. 61/207,484, filed Feb. 12, 2009, theentire contents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to ROS kinase proteins and genesinvolved in cancer, and to the detection, diagnosis and treatment ofcancer.

BACKGROUND OF THE INVENTION

Many cancers are characterized by disruptions in cellular signalingpathways that lead to aberrant control of cellular processes, or touncontrolled growth and proliferation of cells. These disruptions areoften caused by changes in the activity of particular signalingproteins, such as kinases.

It is known that gene translocations resulting in kinase fusion proteinswith aberrant signaling activity can directly lead to certain cancers.For example, it has been directly demonstrated that the BCR-ABLoncoprotein, a tyrosine kinase fusion protein, is the causative agentand drives human chronic myelogenous leukemia (CML). The BCR-ABLoncoprotein, which is found in at least 90-95% of CML cases, isgenerated by the translocation of gene sequences from the c-ABL proteintyrosine kinase on chromosome 9 into BCR sequences on chromosome 22,producing the so-called Philadelphia chromosome. See, e.g. Kurzock etal., N. Engl. J. Med. 319: 990-998 (1988). The translocation is alsoobserved in acute lymphocytic leukemia and AML cases.

Gene translocations leading to mutant or fusion proteins implicated in avariety of other cancers have been described. For example, Falini etal., Blood 99(2): 409-426 (2002), review translocations known to occurin hematological cancers.

Identifying translocations and mutations in human cancers is highlydesirable because it can lead to the development of new therapeuticsthat target such fusion or mutant proteins, and to new diagnostics foridentifying patients that have such gene translocations. For example,BCR-ABL has become a target for the development of therapeutics to treatleukemia. Most recently, Gleevec® (Imatinib mesylate, STI-571), a smallmolecule inhibitor of the ABL kinase, has been approved for thetreatment of CML. This drug is the first of a new class ofanti-proliferative agents designed to interfere with the signalingpathways that drive the growth of tumor cells. The development of thisdrug represents a significant advance over the conventional therapiesfor CML and ALL, chemotherapy and radiation, which are plagued by wellknown side-effects and are often of limited effect since they fail tospecifically target the underlying causes of the malignancies. Likewise,reagents and methods for specifically detecting BCR-ABL fusion proteinin patients, in order to identify patients most likely to respond totargeted inhibitors like Gleevec®, have been described.

Accordingly, there remains a need for the identification of genetranslocations or mutations resulting in fusion or mutant proteinsimplicated in the progression of human cancers, and the development ofnew reagents and methods for the study and detection of such fusionproteins. Identification of such fusion proteins will, among otherthings, desirably enable new methods for selecting patients for targetedtherapies, as well as for the screening of new drugs that inhibit suchmutant/fusion proteins.

SUMMARY OF THE INVENTION

The invention provides a gene translocation involving the ROS kinasegene in human cancer, such as liver, kidney, pancreatic, and testicularcancers (including cancers in the ducts of these tissues, such as bileduct liver cancer), which results in fusion proteins combining part ofthe FIG protein (a Golgi apparatus protein) with the kinase domain ofthe ROS kinase. The FIG-ROS fusion proteins (namely, FIG-ROS(S),FIG-ROS(L), and FIG-ROS(XL)) retain ROS tyrosine kinase activity. Theinvention also provides methods of detection and treatment of humancancers such as liver, kidney, pancreatic, and testicular cancers(including cancers in the ducts of these tissues, such as bile ductliver cancer), which arise not only from gene translocations involvingthe ROS kinase, but also from aberrant expression of the ROS kinase inthese tissues. The invention also provides a truncated ROS kinasewhereby the kinase domain (with or without the transmembrane domain) ofthe ROS kinase is active but separated from the rest of the full-lengthROS kinase (e.g., separate from the extracellular domain of the ROSprotein). The expression of a mutant ROS kinase with active kinaseactivity may drive the proliferation and survival of liver, pancreatic,kidney, and testicular cancers in a subset of such cancers in which atruncated ROS kinase with active kinase activity is expressed.

Accordingly, in a first aspect, the invention provides a purifiedFIG-ROS fusion polypeptide. In some embodiments, the FIG-ROS fusionpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4.In some embodiments, the FIG-ROS fusion polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 2. In some embodiments, theFIG-ROS fusion polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 17. In some embodiments, the FIG-ROS fusion polypeptide isencoded by the nucleic acid sequence set forth in SEQ ID NO: 3. In someembodiments, the FIG-ROS fusion polypeptide is encoded by the nucleicacid sequence set forth in SEQ ID NO: 1. In some embodiments, theFIG-ROS fusion polypeptide is encoded by the nucleic acid sequence setforth in SEQ ID NO: 16.

In a further aspect, the invention provides a purified FIG-ROS fusionpolynucleotide. In some embodiments, the FIG-ROS fusion polynucleotidecomprises the nucleotide sequence set forth in SEQ ID NO: 3. In someembodiments, the FIG-ROS fusion polynucleotide comprises the nucleotidesequence set forth in SEQ ID NO: 1. In some embodiments, the FIG-ROSfusion polynucleotide comprises the nucleotide sequence set forth in SEQID NO: 16.

In another aspect, the invention provides a binding agent thatspecifically binds to a FIG-ROS fusion polypeptide. In some embodiments,the binding agent specifically binds to a fusion junction between a FIGportion and a ROS portion in said FIG-ROS fusion polypeptide. In someembodiments, the fusion junction comprises an amino acid sequenceselected from the group consisting of AGSTLP, LQVWHR, and LQAGVP. Insome embodiments, the FIG-ROS fusion polypeptide is a FIG-ROS(S) fusionpolypeptide, is a FIG-ROS (XL) fusion polypeptide, or is a FIG-ROS (L)fusion polypeptide. In some embodiments, the binding agent is anantibody and an AQUA peptide. In some embodiments, the AQUA peptidecomprises an amino acid sequence selected from the group consisting ofAGSTLP, LQVWHR, and LQAGVP.

In yet another aspect, the invention provides a nucleotide probe fordetecting a FIG-ROS fusion polynucleotide, wherein said probe hybridizesto said FIG-ROS fusion polynucleotide under stringent conditions. Insome embodiments, the FIG-ROS fusion polynucleotide comprises thenucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, theFIG-ROS fusion polynucleotide encodes a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 4. In some embodiments, theFIG-ROS fusion polynucleotide comprises the nucleotide sequence setforth in SEQ ID NO: 1. In some embodiments, the FIG-ROS fusionpolynucleotide encodes a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 2 In some embodiments, the FIG-ROS fusionpolynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:16. In some embodiments, the FIG-ROS fusion polynucleotide encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:17.

In another aspect, the invention provides a method for detecting aFIG-ROS gene translocation, the method comprising contacting abiological sample with a binding agent that specifically binds to aFIG-ROS fusion polypeptide (e.g., a FIG-ROS(S), FIG-ROS(XL), or aFIG-ROS(L) fusion polypeptide), where specific binding of the bindingagent to the biological sample indicates the presence of a FIG-ROS genetranslocation (e.g., that encodes a FIG-ROS(S), FIG-ROS(XL), orFIG-ROS(L) fusion polypeptide) in said biological sample.

In a further aspect, the invention provides a method for detecting aFIG-ROS gene translocation by contacting a biological sample with anucleotide probe that hybridizes to a FIG-ROS fusion polynucleotideunder stringent conditions, wherein hybridization of said nucleotideprobe to said biological sample indicates a FIG-ROS gene translocation(e.g., that encodes a FIG-ROS(S), FIG-ROS(XL) or FIG-ROS(L) fusionpolypeptide) in said biological sample.

In yet another aspect, the invention provides a method for diagnosing apatient as having a cancer or a suspected cancer characterized by a ROSkinase. In some embodiments, the cancer or suspected cancer is notnon-small cell lung carcinoma or glioblastoma. The method includescontacting a biological sample of said cancer or suspected cancer (wherethe biological sample comprising at least one polypeptide) with abinding agent that specifically binds to a mutant ROS polypeptide,wherein specific binding of said binding agent to at least onepolypeptide in said biological sample identifies said patient as havinga cancer or a suspected cancer characterized by a ROS kinase.

In another aspect, the invention provides a method for identifying acancer (or a suspected cancer) that is likely to respond to a ROSinhibitor. In some embodiments, the cancer or suspected cancer is notnon-small cell lung carcinoma or glioblastoma. The method includescontacting a biological sample of said cancer (or suspected cancer)comprising at least one polypeptide, with a binding agent thatspecifically binds to a mutant ROS polypeptide, wherein specific bindingof said binding agent to at least one polypeptide in said biologicalsample identifies said cancer or suspected cancer as a cancer orsuspected cancer that is likely to respond to a ROS inhibitor.

In various embodiments, the mutant ROS polypeptide is aberrantlyexpressed wild-type ROS polypeptide. For example, aberrant expressioncan be where wild-type ROS kinase is overexpressed in a cancer or asuspected cancer as compared to the level of expression of wild-type ROSkinase in normal tissue of the same tissue type as the cancer orsuspected cancer. ROS protein expression levels can be determined bystandard means (e.g., Western blotting analysis, mass spectrometry, IHCstaining).

In various embodiments, the mutant ROS polypeptide is a truncated ROSpolypeptide or a ROS fusion polypeptide. Non-limiting examples of ROSfusion polypeptides include a FIG-ROS(S) fusion polypeptide, aFIG-ROS(L) fusion polypeptide, a FIG-ROS(XL) fusion polypeptide, aSLC34A2-ROS(S) fusion polypeptide, a SLC34A2-ROS(L) fusion polypeptide,a SLC34A2-ROS(VS) fusion polypeptide, and a CD74-ROS fusion polypeptide.Non-limiting examples of a truncated ROS polypeptide include the kinasedomain of ROS lacking the extracellular and transmembrane domains ofwild-type ROS and the transmembrane and kinase domains of ROS lackingthe extracellular domain of wild-type ROS.

In some embodiments, the binding agent is an antibody or an AQUApeptide. In some embodiments, the cancer is from a patient (e.g., ahuman patient).

In a further aspect, the invention provides a method for diagnosing apatient as having a cancer or a suspected cancer characterized by a ROSkinase. In some embodiments, the cancer or suspected cancer is notnon-small cell lung carcinoma or glioblastoma. The method includescontacting a biological sample of said cancer or a suspected cancer(where the biological sample comprising at least one nucleic acidmolecule) with a probe that hybridizes under stringent conditions to anucleic acid molecule selected from the group consisting of a FIG-ROSfusion polynucleotide, a SLC34A2-ROS fusion polypeptide, a CD74-ROSfusion polypeptide, and a truncated ROS polynucleotide, and whereinhybridization of said probe to at least one nucleic acid molecule insaid biological sample identifies said patient as having a cancer or asuspected cancer characterized by a ROS kinase.

In yet another aspect, the invention provides another method foridentifying a cancer (or suspected cancer) that is likely to respond toa ROS inhibitor. The method includes contacting a biological sample ofsaid cancer comprising at least one nucleic acid molecule with anucleotide probe that hybridizes under stringent conditions to a eithera FIG-ROS fusion polynucleotide (e.g., a FIG-ROS(S) or FIG-ROS(L) fusionpolynucleotide) or a mutant ROS polynucleotide, and whereinhybridization of said nucleotide probe to at least one nucleic acidmolecule in said biological sample identifies said cancer as a cancerthat is likely to respond to a ROS inhibitor. In some embodiments, theFIG-ROS fusion polynucleotide encodes a FIG-ROS(S) fusion polypeptide, aFIG-ROS(L) fusion polypeptide, or a FIG-ROS(XL) fusion polypeptide. Insome embodiments, the SCL34A2-ROS fusion polynucleotide encodes aSCL34A2-ROS(S) fusion polypeptide, a SCL34A2-ROS(L) fusion polypeptide,or a SCL34A2-ROS(VS) fusion polypeptide. In some embodiments, the canceris from a patient (e.g., a cancer patient). In some embodiments, thepatient is human.

In various embodiments of all aspects of the invention, the cancer maybe a liver cancer, a pancreatic cancer, a kidney cancer, or a testicularcancer. In various embodiments, the cancer may be a duct cancer (e.g., aliver bile duct cancer or a pancreatic duct cancer). In furtherembodiments, the cancer is not a non-small cell lung cancer (NSCLC). Infurther embodiments, the cancer is not a glioblastoma). In furtherembodiments, the ROS inhibitor also inhibits the activity of an ALKkinase an LTK kinase, an insulin receptor, or an IGF1 receptor. Infurther embodiments, the ROS inhibitor is PF-02341066 or NVP-TAE684).

In further embodiments, the ROS inhibitor is a binding agent thatspecifically binds to a FIG-ROS fusion polypeptide, a binding agent thatspecifically binds to a truncated ROS polypeptide, an siRNA targeting aFIG-ROS fusion polynucleotide, or an siRNA targeting a truncated ROSpolynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the location of the FIG gene and ROS gene on chromosome 6.Both FIG and ROS genes are localized on chromosome 6q22.2 with about 0.2Mega base pairs apart. The FIG gene is also known as the GOPC gene.

FIG. 2 shows the breakpoint in the FIG and the ROS proteins, forming twoFIG-ROS fusion proteins. The FIG-ROS (L) fusion protein results frombreaks in the Fig and the Ros genes at the black arrows, while theFIG-ROS(S) fusion protein results from breaks in the Fig and the Rosgenes at the red arrows.

FIG. 3 is a depiction of an agarose gel showing the detection of the twofusion gene transcripts, FIG-ROS(S) and FIG-ROS(L) formed by the FIG andROS translocation by RT-PCR in the liver cancer samples from twopatients, namely XY3-78T and 090665LC.

FIG. 4 is a depiction of an agarose gel showing the expression ofwild-type FIG, wild-type ROS, and the FIG-ROS fusion transcript byRT-PCR in the liver cancer samples from two patients, namely XY3-78T and090665LC. The U118MG human glioblastoma cell line, which has aFIG-ROS(L) translocation, is also shown. HCC78 a human non-small celllung cancer cell line, which contains SLC34A2-ROS translocation, wasserved as a negative control.

FIG. 5 is a depiction of an agarose gel showing the PCR productsgenerated by amplifying genomic DNA from liver cancer samples frompatients XY3-78T and 090665LC, and from cell line U118MG.

FIG. 6 is a depiction of a Western blotting analysis showing theexpression of FIG-ROS(S) from XY3-78T, FIG-ROS(L) from 090665LC, andFIG-ROS(L) from U118MG cells.

FIG. 7 is a photograph of four tissue culture plates containing 3t3cells cultured in soft agar, where the 3T3 cells are stably transfectedwith FIG-ROS(L) (upper left), FIG-ROS(S) (upper right), src kinase(lower left) and empty vector (lower right).

FIG. 8 is a photograph showing nude mice injected with 3T3 cells stablytransfected with empty vector (left), FIG-ROS(L) (middle), orFIG-ROS(S).

FIGS. 9A and 9B are photographs of cells showing the subcellularlocalization of FIG-ROS(L) and FIG-ROS(S) in 3T3 cells.

FIG. 10 is a depiction of a Western blotting analysis showing the stableexpression of FIG-ROS(S), FIG-ROS(L), and FIG-ROS(L) from U118MG in BaF3cells grown with or without IL-3.

FIG. 11 is a line graph showing the ability of BAF3 cells transducedwith retrovirus encoding FIG-ROS(S) (red squares) or FIG-ROS(L) (bluediamonds) to grow without the presence of IL-3. BAF3 cells transducedwith empty retrovirus is also shown (light purple line).

FIG. 12 is a bar graph showing the results of an in vitro kinase assay(top) made by quantitating the bands on the gel (below) from BaF3 cellstransduced with retrovirus encoding FIG-ROS(S), FIG-ROS(L) or emptyvirus (“neo”).

FIG. 13 is a line graph showing the cellular growth response in thepresence of 0 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM or 1000 nM TAE-684of BaF3 expressing FIG-ROS(S) (red squares), BaF3 expressing FIG-ROS(L)(blue diamonds), BaF3 expressing FLT31TD (green triangles), and Karpas299 cells (purple Xs).

FIG. 14 is a bar graph showing that BaF3 expressing either FIG-ROS(S) orFIG-ROS(L) die by apoptosis in the presence of TAE-684.

FIG. 15 is a depiction of a Western blotting analysis showing thatphosphorylation of both FIG-ROS(S) and FIG-ROS(L), as well as theirdownstream signaling molecules, are inhibited by TAE-684.

FIG. 16 is a schematic representation of the various BAC clones thathybridize to the FIG and ROS genes.

FIG. 17 is an image of an IHC slide from a representative, non-limitingCCA tissue sample that stained positive for ROS expression.

FIG. 18 is an image of an IHC slide from a representative, non-limitingHCC tissue sample that stained moderately positive for ROS expression.

FIGS. 19A and 9B are images of representative, non-limiting IHC slidesstained with the ROS-specific antibody following the addition of peptideROS-1 (FIG. 19A) and peptide ROS-9 (FIG. 19B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides a mutant ROS kinase which is expressed in asubset of human liver, kidney, pancreatic, and testicular cancers (e.g.,bile duct liver cancer). The mutant ROS kinase may drive theproliferation and survival of liver, pancreatic, kidney, and testicularcancers in a subset of such cancers in which the mutant ROS kinase isexpressed.

The published patents, patent applications, websites, company names, andscientific literature referred to herein establish the knowledge that isavailable to those with skill in the art and are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter.

The further aspects, advantages, and embodiments of the invention aredescribed in more detail below. The patents, published applications, andscientific literature referred to herein establish the knowledge ofthose with skill in the art and are hereby incorporated by reference intheir entirety to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Any conflictbetween any reference cited herein and the specific teachings of thisspecification shall be resolved in favor of the latter. Likewise, anyconflict between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification shall be resolved in favor of the latter. As used herein,the following terms have the meanings indicated. As used in thisspecification, the singular forms “a,” “an” and “the” specifically alsoencompass the plural forms of the terms to which they refer, unless thecontent clearly dictates otherwise. The term “about” is used herein tomean approximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies thatrange by extending the boundaries above and below the numerical valuesset forth. In general, the term “about” is used herein to modify anumerical value above and below the stated value by a variance of 20%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989);Kaufman et al., Eds., Handbook of Molecular and Cellular Methods inBiology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed.,Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).Standard reference works setting forth the general principles ofpharmacology include Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).

The invention relates to the discovery of mutant ROS (i.e., aberrantlyexpressed full length ROS, truncated (i.e., less than full length) ROS,or ROS fusion proteins (e.g., the FIG-ROS fusions, the SLC34A2-ROSfusions, or the CD74-ROS fusion)) in liver cancer (including bile ductcancer), pancreatic cancer, kidney cancer, and testicular cancer. Theinvention further relates to the discovery of new ROS genetranslocations, resulting in fusions between the FIG gene and the ROSgene.

Full length (wild-type) ROS kinase is a 2347 amino acid long receptortyrosine kinase. In humans, ROS kinase RNA has been detected inplacenta, lung and skeletal muscle, with possible low levels ofexpression in testes (see J. Acquaviva, et al., Biochim. Biophys. Acta1795(1):37-52, 2009. However, full-length ROS kinase does not appear tobe expressed in normal liver, kidney, and pancreas tissue in humans (seeJ. Acquaviva, et al., supra). While Abcam Inc. (Cambridge, Mass.) sellsa ROS-specific antibody (clone ab5512) that allegedly stains (i.e.,specifically binds to) human hepatocarcinoma tissue by IHC, this ab5512was found to stain paraffin-embedded HCC78 cells (lung carcinoma whichexpress ROS) and HCC827 cells (lung adenocarcinoma which do not expressROS) with equal intensity (cells obtained from the ATCC, data notshown). Additionally, although ROS kinase may be present in humantesticular tissue, its expression appears to be limited to theepididymis (see Acquaviva, et al., supra).

Accordingly, in a first aspect, the invention provides a purifiedFIG-ROS fusion polypeptide. By “FIG-ROS fusion polypeptide” is meant theFIG-ROS fusion polypeptide (e.g., FIG-ROS(L), FIG-ROS(XL), orFIG-ROS(S)) described herein, obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, and human,from any source whether natural, synthetic, semi-synthetic, orrecombinant.

By “purified” (or “isolated″”) refers to a nucleic acid sequence (e.g.,a polynucleotide) or an amino acid sequence (e.g., a polypeptide) thatis removed or separated from other components present in its naturalenvironment. For example, an isolated FIG-ROS fusion polypeptide is onethat is separated from other components of a eukaryotic cell (e.g., theendoplasmic reticulum or cytoplasmic proteins and RNA). An isolatedFIG-ROS polynucleotide is one that is separated from other nuclearcomponents (e.g., histones) and/or from upstream or downstream nucleicacid sequences (e.g., an isolated FIG-ROS polynucleotide is separatedfrom the endogenous FIG gene promoter). An isolated nucleic acidsequence of amino acid sequence of the invention is at least 60% free,or at least 75% free, or at least 90% free, or at least 95% free fromother components present in natural environment of the indicated nucleicacid sequence or acid sequence.

A FIG-ROS fusion polypeptide of the invention is a non-limiting exampleof mutant ROS polypeptide.

As used herein, the term “mutant ROS” polypeptide or polynucleotidemeans either the aberrant expression of the wild-type ROS kinasepolypeptide or polynucleotide in a tissue in which ROS kinase is notnormally expressed (or expressed at a different level) or the kinasedomain of a ROS or a polynucleotide encoding the kinase domain of a ROSkinase without the extracellular domain or without the transmembranedomains of wild-type (i.e., full length) ROS, where the kinase domain(with or without the transmembrane domain) is either alone (alsoreferred to as truncated ROS) or is fused to all or a portion of asecond protein (e.g., a FIG protein).

Wild-type ROS kinase is a 2347 amino acid long receptor tyrosine kinase,where approximately the first 36 amino acids (i.e., the N-terminal 36amino acids) are the signal peptide. The sequence of human ROS kinasecan be found at GenBank Accession No. M34353, and the protein sequence(including the signal peptide) is provided herein as SEQ ID NO: 9.

Non-limiting examples of the mutant ROS polypeptide of the inventioninclude polypeptides comprising the amino acid sequences set forth inSEQ ID NO: 12 or SEQ ID NO: 13. Likewise, in certain embodiments,non-limiting examples of mutant ROS polynucleotides of the inventioninclude polynucleotides encoding polypeptides comprising the amino acidsequences set forth in SEQ ID NO: 12 or SEQ ID NO: 13. In someembodiments, the mutant ROS polynucleotide comprises a portion of thenucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO:7, or SEQ ID NO: 8. In certain embodiments, the mutant ROSpolypeptide of the invention does not include the sequences of SEQ IDNO: 10 or SEQ ID NO: 11. Likewise, in certain embodiments, non-limitingexamples of mutant ROS polynucleotides of the invention do not includepolynucleotides encoding polypeptides comprising the amino acidsequences set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

Thus, a mutant ROS comprises the kinase domain, with or without thetransmembrane domain, of ROS (or nucleotide sequences encoding the same)such that the kinase domain of the ROS kinase (with or without thetransmembrane domain) is separated from the other domains (e.g., theextracellular domain) of wild-type (i.e., full-length) ROS kinase. Thefull length amino acid sequence of ROS kinase is provided in SEQ ID NO:9. The kinase domain of the ROS kinase is provided in SEQ ID NOs: 12 and13; however the term “mutant ROS” includes also those amino acidresidues which flank the kinase domain provided that the flanking aminoacid residues are not within the transmembrane domain or extracellulardomain of the full-length ROS protein. In some embodiments, the mutantROS excludes the sequence set forth in SEQ ID NO: 11. In someembodiments, the mutant ROS excludes the sequence set forth in SEQ IDNO: 10. Thus, the mutant ROS described herein includes the amino acidsequence set forth in SEQ ID NO: 3 and a nucleotide sequence encodingthe same. The term “mutant ROS polypeptide” also includes a chimericprotein that includes all or part of a second protein fused by a peptidebond to the kinase domain of a ROS polypeptide. As discussed above, onenon-limiting example of a mutant ROS polypeptide that is a chimericprotein is the FIG-ROS(S) fusion polypeptide described herein. Likewise,the term “mutant ROS polynucleotide also includes a polynucleotideencoding a chimeric protein that includes all or part of a secondprotein fused by a peptide bond to the kinase domain of a ROSpolypeptide.

Thus, as used herein, the term mutant ROS includes, without limitation,the FIG-ROS (L) fusion polypeptide (see nucleic acid sequence in SEQ IDNO: 1 and amino acid sequence in SEQ ID NO: 2), the FIG-ROS(S) fusionpolypeptide (see nucleic acid sequence in SEQ ID NO: 3 and amino acidsequence in SEQ ID NO: 4), the FIG-ROS(XL) fusion polypeptide (seenucleic acid sequence in SEQ ID NO: 16 and amino acid sequence in SEQ IDNO: 17), the SLC34A2-ROS (L) fusion polypeptide (see nucleic acidsequence in SEQ ID NO: 18 and amino acid sequence in SEQ ID NO: 19), theSLC34A2-ROS(S) fusion protein (see nucleic acid sequence in SEQ ID NO:20 and amino acid sequence in SEQ ID NO: 21), the SLC34A2-ROS (VS)fusion protein (see nucleic acid sequence in SEQ ID NO: 22 and aminoacid sequence in SEQ ID NO: 23), and the CD74-ROS fusion protein (seenucleic acid sequence in SEQ ID NO: 24 and amino acid sequence in SEQ IDNO: 25). Note that additional ROS fusion polypeptides are disclosed inPCT Publication No. WO2007084631; Rikova, K et al., Cell 131:1190-1203,2007, and PCT Publication No. WO/2009/051846, the entire contents ofwhich are hereby incorporated by reference.

As used herein, by “polynucleotide” (or “nucleotide sequence” or“nucleic acid molecule”) refers to an oligonucleotide, nucleotide, orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand.

As used herein, by “polypeptide” (or “amino acid sequence” or protein)refers to an oligopeptide, peptide, polypeptide, or protein sequence,and fragments or portions thereof, and to naturally occurring orsynthetic molecules. “Amino acid sequence” and like terms, such as“polypeptide” or “protein”, are not meant to limit the indicated aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

In accordance with the invention, human FIG-ROS gene translocation havebeen identified using global phosphopeptide profiling in liver cancersamples taken from human patients (see Examples below). These genetranslocations which occurs on human chromosome (6q22) result inexpression of two variant fusion proteins, namely the FIG-ROS(S) fusionpolypeptide and the FIG-ROS(L) fusion polypeptide) that combine theN-terminus of FIG with the kinase domain of ROS.

As used herein, by “cancer” or “cancerous” is meant a cell that showsabnormal growth as compared to a normal (i.e., non-cancerous) cell ofthe same cell type. For example, a cancerous cell may be metastatic ornon-metastatic. A cancerous cell may also show lack of contactinhibition where a normal cell of that same cell type shows contactinhibition. As used herein, by “suspected cancer” or “tissue suspectedof being cancerous” is meant a cell or tissue that has some aberrantcharacteristics (e.g., hyperplastic or lack of contact inhibition) ascompared to normal cells or tissues of that same cell or tissue type asthe suspected cancer, but where the cell or tissue is not yet confirmedby a physician or pathologist as being cancerous.

As shown in FIGS. 1 and 2, the FIG-ROS(L) translocation combines thenucleic acid sequence encoding the N-terminus of FIG (amino acids 1-412)with the nucleic acid sequences encoding the kinase domain of ROS (aminoacids 413-878 which correspond to amino acids 1882-2347 from ROS) (seeSEQ ID NO: 2), to produce a fusion, namely FIG-ROS(L) fusionpolypeptide. The resulting FIG-ROS(L) fusion protein, which comprises878 amino acids, was found to retain the kinase activity of ROS. In someembodiments, the FIG-ROS fusion polypeptide is a FIG-ROS(L) fusionpolypeptide. In some embodiments, the FIG-ROS (L) fusion polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 2. In someembodiments, the FIG-ROS (L) fusion polypeptide is encoded by thenucleic acid sequence set forth in SEQ ID NO: 1.

Also shown in FIGS. 1 and 2, the FIG-ROS(S) translocation combines thenucleic acid sequence encoding the N-terminus of FIG (amino acids 1-209)with the nucleic acid sequence encoding the kinase domain of ROS (aminoacids 210-630 which correspond to amino acids 1927-2347 from ROS) (seealso SEQ ID NO:4), to produce a fusion, namely the FIG-ROS(S) fusionpolypeptide. The resulting FIG-ROS(S) fusion protein, which comprises630 amino acids, was found to retain the kinase activity of ROS. Thus,in some embodiments, the FIG-ROS fusion polypeptide of the invention isa FIG-ROS(S) fusion polypeptide. In some embodiments, the FIG-ROS(S)fusion polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 4. In some embodiments, the FIG-ROS(S) fusion polypeptide is encodedby the nucleic acid sequence set forth in SEQ ID NO: 3.

The invention further provides a third FIG-ROS fusion, namelyFIG-ROS(XL), which translocation combines the nucleic acid sequenceencoding the N-terminus of FIG (amino acids 1-411 or 1-412) with thenucleic acid sequences encoding the transmembrane and kinase domains ofROS kinase to result in a fusion protein of 1009 amino acids in length.

It should be noted that in all of the ROS fusion proteins describedherein (e.g., the FIG-ROS fusion proteins, the SLC34A2-ROS fusionproteins, and the CD74-ROS fusion protein), the amino acid at the fusionjunction (regardless of the numbering) may appear in either wild-typeprotein member of the fusion (e.g., the amino acid at the fusionjunction in a FIG-ROS fusion polypeptide may appear in either wild-typeFIG protein or wild-type ROS protein), or the amino acid, being createdby a codon with nucleotides from fused exons of both protein members,may be unique to the fusion polypeptide and not appear in eitherwild-type protein member of the fusion.

The invention provides that mutant ROS may be present liver cancer(including bile duct cancer), kidney cancer, testicular cancer, andpancreatic cancer. Based on these discoveries, patients suffering fromthese cancers whose cancers express mutant ROS (e.g., over-expresswild-type ROS or express a truncated ROS or a ROS fusion polypeptidesuch as one of the FIG-ROS fusion polypeptides disclosed herein) mayrespond favorably to administration of a ROS inhibitor (e.g., the growthof their cancer may slow or stop as compared to untreated patientssuffering from the same cancer).

Thus, the invention provides isolated FIG-ROS fusion polypeptides andfragments thereof. In one embodiment, the invention provides an isolatedpolypeptide comprising an amino acid sequence at least 95% identical orat least 99% identical to a sequence selected from the group consistingof: (a) an amino acid sequence encoding a FIG-ROS fusion polypeptidecomprising the amino acid sequence of SEQ ID NO: 1; (b) an amino acidsequence encoding a FIG-ROS fusion polypeptide comprising the amino acidsequence of SEQ ID NO: 17; (c) an amino acid sequence encoding a FIG-ROSfusion polypeptide comprising all or a portion of the FIG polypeptidewith the kinase domain of ROS (e.g., SEQ ID NO: 12 or 13)); and (d) anamino acid sequence encoding a polypeptide comprising at least sixcontiguous amino acids encompassing the fusion junction of a FIG-ROSfusion polypeptide (e.g., AGSTLP of FIG-ROS (S), LQVWHR of FIG-ROS(L),or LQAGVP of FIG-ROS(XL)).

In one embodiment, the invention provides an isolated FIG-ROS(S) fusionpolypeptide having the amino acid sequence set forth in SEQ ID NO: 4. Inone embodiment, the invention provides an isolated FIG-ROS (XL) fusionpolypeptide having the amino acid sequence set forth in SEQ ID NO: 17.In another embodiment, recombinant mutant polypeptides of the inventionare provided, which may be produced using a recombinant vector orrecombinant host cell as described above.

It will be recognized in the art that some amino acid sequences of aFIG-ROS fusion polypeptide can be varied without significant effect ofthe structure or function of the mutant protein. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity (e.g. the kinasedomain of ROS). In general, it is possible to replace residues that formthe tertiary structure, provided that residues performing a similarfunction are used. In other instances, the type of residue may becompletely unimportant if the alteration occurs at a non-critical regionof the protein.

Thus, the invention further includes a FIG-ROS variant of a FIG-ROSfusion polypeptide that shows substantial ROS kinase activity or thatincludes regions of FIG and ROS proteins. In some embodiments, a FIG-ROSvariant of the invention contains conservative substitutions as comparedto FIG-ROS(L), FIG-ROS (XL), or FIG-ROS(S). Some non-limitingconservative substitutions include the exchange, one for another, amongthe aliphatic amino acids Ala, Val, Leu and Ile; exchange of thehydroxyl residues Ser and Thr; exchange of the acidic residues Asp andGlu; exchange of the amide residues Asn and Gln; exchange of the basicresidues Lys and Arg; and exchange of the aromatic residues Phe and Tyr.Further examples of conservative amino acid substitutions known to thoseskilled in the art are: Aromatic: phenylalanine tryptophan tyrosine(e.g., a tryptophan residue is replaced with a phenylalanine);Hydrophobic: leucine isoleucine valine; Polar: glutamine asparagines;Basic: arginine lysine histidine; Acidic: aspartic acid glutamic acid;Small: alanine serine threonine methionine glycine. As indicated indetail above, further guidance concerning which amino acid changes arelikely to be phenotypically silent (i.e., are not likely to have asignificant deleterious effect on a function) can be found in Bowie etal., Science 247, supra.

In some embodiments, a variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Similar variants may alsoinclude amino acid deletions or insertions, or both. Guidance indetermining which amino acid residues may be substituted, inserted, ordeleted without abolishing biological or immunological activity may befound using computer programs well known in the art, for example,DNASTAR software.

The FIG-ROS fusion polypeptides, fragments thereof, and variants thereofof the present invention may be provided in an isolated or purifiedform. A recombinantly produced version of a FIG-ROS fusion polypeptideof the invention can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67: 31-40 (1988).

The polypeptides of the present invention include the FIG-ROS fusionpolypeptides having the sequences set forth in SEQ ID NOs: 2 and 4, and17 (whether or not including a leader sequence), an amino acid sequenceencoding a polypeptide comprising at least six contiguous amino acidsencompassing the fusion junction of a FIG-ROS fusion polypeptide of theinvention, as well as polypeptides that have at least 90% similarity,more preferably at least 95% similarity, and still more preferably atleast 96%, 97%, 98% or 99% similarity to those described above.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981))to find the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a mutant ROSpolypeptide of the invention is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid sequence of the FIG-ROSfusion polypeptide. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

When using Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

A FIG-ROS fusion polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns, for example, using methods well known to those ofskill in the art.

As further described in detail below, the polypeptides of the presentinvention can also be used to generate fusion polypeptide specificreagents, such as polyclonal and monoclonal antibodies, which are usefulin assays for detecting mutant ROS polypeptide expression as describedbelow or as agonists and antagonists capable of enhancing or inhibitingmutant ROS protein function/activity. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” FIG-ROS fusionpolypeptide binding proteins, which are also candidate agonist andantagonist according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340: 245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention,such as an epitope comprising the fusion junction of a FIG-ROS fusionpolypeptide variant An “epitope” refers to either an immunogenic epitope(i.e., capable of eliciting an immune response) or an antigenic epitope(i.e., the region of a protein molecule to which an antibody canspecifically bind. The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).The production of FIG-ROS fusion polypeptide-specific antibodies of theinvention is described in further detail below.

The antibodies that specifically bind to an epitope-bearing peptides orpolypeptides are useful to detect a mimicked protein, and antibodies todifferent peptides may be used for tracking the fate of various regionsof a protein precursor which undergoes post-translational processing.The peptides and anti-peptide antibodies may be used in a variety ofqualitative or quantitative assays for the mimicked protein, forinstance in competition assays since it has been shown that even shortpeptides (e.g., about 9 amino acids) can bind and displace the largerpeptides in immunoprecipitation assays. See, for instance, Wilson etal., Cell 37: 767-778 (1984) at 777. The anti-peptide antibodies of theinvention also are useful for purification of the mimicked protein, forinstance, by adsorption chromatography using methods well known in theart. Immunological assay formats are described in further detail below.

Recombinant mutant ROS kinase polypeptides are also within the scope ofthe present invention, and may be producing using fusion polynucleotidesof the invention, as described above. For example, the inventionprovides a method for producing a recombinant FIG-ROS fusion polypeptideby culturing a recombinant host cell (as described above) underconditions suitable for the expression of the fusion polypeptide andrecovering the polypeptide. Culture conditions suitable for the growthof host cells and the expression of recombinant polypeptides from suchcells are well known to those of skill in the art.

In a further aspect, the invention provides a purified FIG-ROS fusionpolynucleotide. By “FIG-ROS fusion polynucleotide” or “FIG-ROSpolynucleotide” is meant a FIG-ROS translocation gene (i.e., a gene thathas undergone translocation) or polynucleotide encoding a FIG-ROS fusionpolypeptide (e.g., the FIG-ROS(L), FIG-ROS (XL), or FIG-ROS(S)) fusionpolypeptides described herein), obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, and human,from any source whether natural, synthetic, semi-synthetic, orrecombinant.

In some embodiments, the FIG-ROS fusion polynucleotide comprises thenucleotide sequence set forth in SEQ ID NO:1. In some embodiments, theFIG-ROS fusion polynucleotide encodes a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 2. In some embodiments, theFIG-ROS fusion polynucleotide comprises the nucleotide sequence setforth in SEQ ID NO:3. In some embodiments, the FIG-ROS fusionpolynucleotide encodes a polypeptide having the amino acid sequence setforth in SEQ ID NO: 4. In some embodiments, the FIG-ROS fusionpolynucleotide comprises the nucleotide sequence set forth in SEQ IDNO:16. In some embodiments, the FIG-ROS fusion polynucleotide encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO: 17.

In some embodiments, the FIG-ROS fusion polynucleotide comprises aportion of the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ IDNO: 26. As used herein, a “portion” or “fragment” means a sequencefragment less than the whole sequence (e.g., a 50 nucleotide sequence isa portion of a 100 nucleotide long sequence). In other words, theFIG-ROS fusion polynucleotide may comprise portions of intron sequencesthat do not encode any amino acids in the resulting FIG-ROS fusionpolypeptide.

Thus, the present invention provides, in part, isolated polynucleotidesthat encode a FIG-ROS fusion polypeptide of the invention, nucleotideprobes that hybridize to such polynucleotides, and methods, vectors, andhost cells for utilizing such polynucleotides to produce recombinantfusion polypeptides. Unless otherwise indicated, all nucleotidesequences determined by sequencing a DNA molecule herein were determinedusing an automated DNA sequencer (such as the Model 373 from AppliedBiosystems, Inc.), and all amino acid sequences of polypeptides encodedby DNA molecules determined herein were determined using an automatedpeptide sequencer. As is known in the art for any DNA sequencedetermined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical, andmore typically at least about 95% to about 99.9% identical to the actualnucleotide sequence of the sequenced DNA molecule. The actual sequencecan be more precisely determined by other approaches including manualDNA sequencing methods well known in the art. As is also known in theart, a single insertion or deletion in a determined nucleotide sequencecompared to the actual sequence will cause a frame shift in translationof the nucleotide sequence such that the predicted amino acid sequenceencoded by a determined nucleotide sequence will be completely differentfrom the amino acid sequence actually encoded by the sequenced DNAmolecule, beginning at the point of such an insertion or deletion.Unless otherwise indicated, each nucleotide sequence set forth herein ispresented as a sequence of deoxyribonucleotides (abbreviated A, G, C andT). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO: 3 or set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of SEQ ID NO: 3 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

In one embodiment, the invention provides an isolated polynucleotidecomprising a nucleotide sequence at least about 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a FIG-ROS fusion polypeptide comprising the amino acidsequence of SEQ ID NO: 4 (FIG-ROS(S)); (b) a nucleotide sequenceencoding a FIG-ROS fusion polypeptide comprising the amino acid sequenceof SEQ ID NO: 17 (FIG-ROS(XL)); (c) a nucleotide sequence comprising atleast six contiguous nucleotides encompassing the fusion junction of aFIG-ROS(S) fusion polynucleotide (e.g., AAGTAC), a nucleotide sequencecomprising at least six contiguous nucleotides encompassing the fusionjunction of a FIG-ROS(XL) fusion polynucleotide (e.g., AAGctg); (d) anucleotide sequence encoding at least six contiguous amino acid residuesencompassing the fusion junction of a FIG-ROS(S) fusion polypeptide(e.g., AGSTLP), (e) a nucleotide sequence encoding at least sixcontiguous amino acid residues encompassing the fusion junction of aFIG-ROS(XL) fusion polypeptide (e.g., LQAGVP) and (f) a nucleotidesequence complementary to any of the nucleotide sequences of (a), (b),(c), (d), or (e).

Using the information provided herein, such as the nucleotide sequencesset forth in SEQ ID NOs: 1, 3, and 16, a nucleic acid molecule of thepresent invention encoding a FIG-ROS fusion polypeptide of the inventionmay be obtained using standard cloning and screening procedures, such asthose for cloning cDNAs using mRNA as starting material. The fusion genecan also be identified in cDNA libraries in other human cancers in whichthe FIG-ROS translocation occurs, or in which a deletion or alternativetranslocation results in expression of a truncated ROS kinase lackingthe extracellular domain and may additionally lack the transmembranedomain of the wild type ROS kinase.

The determined nucleotide sequence of the FIG-ROS translocation genesencode the FIG-ROS(S) fusion polypeptide, the FIG-ROS(L) fusionpolypeptide, and the FIG-ROS(XL) fusion polypeptide. The FIG-ROS fusionpolynucleotides comprise the portion of the nucleotide sequence of wildtype FIG that encodes the N-terminus of that protein with the portion ofthe nucleotide sequence of wild type ROS that encodes the kinase domainof that protein

As indicated, the present invention provides, in part, the mature formof the FIG-ROS fusion proteins. According to the signal hypothesis,proteins secreted by mammalian cells have a signal or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Most mammalian cells and even insect cells cleave secretedproteins with the same specificity. However, in some cases, cleavage ofa secreted protein is not entirely uniform, which results in two or moremature species on the protein. Further, it has long been known that thecleavage specificity of a secreted protein is ultimately determined bythe primary structure of the complete protein, that is, it is inherentin the amino acid sequence of the polypeptide. Therefore, the presentinvention provides, in part, nucleotide sequences encoding a matureFIG-ROS(S) fusion polypeptide having the nucleotide sequence set forthin SEQ ID NO: 3 with additional nucleic acid residues located 5′ to the5′-terminal residues of SEQ ID NO. 3 and includes the amino acidsequence of a FIG-ROS(S) fusion polypeptide having the amino acidsequence set forth in SEQ ID NO: 4 with additional amino acid residueslocated N-terminally to the N-terminal residue of SEQ ID NO. 4. Theinvention also provides, in part, nucleotide sequences encoding a matureFIG-ROS(XL) fusion polypeptide having the nucleotide sequence set forthin SEQ ID NO: 16 with additional nucleic acid residues located 5′ to the5′-terminal residues of SEQ ID NO. 16 and includes the amino acidsequence of a FIG-ROS(XL) fusion polypeptide having the amino acidsequence set forth in SEQ ID NO: 17 with additional amino acid residueslocated N-terminally to the N-terminal residue of SEQ ID NO. 17.

As indicated, polynucleotides of the present invention may be in theform of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

Isolated polynucleotides of the invention are nucleic acid molecules,DNA or RNA, which have been removed from their native environment. Forexample, recombinant DNA molecules contained in a vector are consideredisolated for the purposes of the present invention. Further examples ofisolated DNA molecules include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules include in vivo or invitro RNA transcripts of the DNA molecules of the present invention.Isolated nucleic acid molecules according to the present inventionfurther include such molecules produced synthetically.

Isolated polynucleotides of the invention include the nucleic acidmolecules having the sequences set forth in (SEQ ID NOs: 1, 3, and 16,nucleic acid molecules comprising the coding sequence for theFIG-ROS(S), FIG-ROS(L), and FIG-ROS(XL) fusion proteins that comprise asequence different from those described above but which, due to thedegeneracy of the genetic code, still a mutant ROS polypeptide of theinvention. The genetic code is well known in the art, thus, it would beroutine for one skilled in the art to generate such degenerate variants.

In another embodiment, the invention provides an isolated polynucleotideencoding the FIG-ROS fusion polypeptide comprising the FIG-ROStranslocation nucleotide sequence contained in the above-described cDNAclones. In some embodiments, such nucleic acid molecule will encode themature FIG-ROS(S) fusion polypeptide, the mature FIG-ROS(L) fusionpolypeptide, or the mature FIG-ROS(XL) fusion polypeptide. In anotherembodiment, the invention provides an isolated nucleotide sequenceencoding a FIG-ROS fusion polypeptide comprising the N-terminal aminoacid sequence of FIG and the kinase domain of ROS. In one embodiment,the polypeptide comprising the kinase domain of ROS comprises the aminoacid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13. In anotherembodiment, the N-terminal amino acid sequence of FIG and kinase domainof ROS are encoded by the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 16.

The invention further provides isolated polynucleotides comprisingnucleotide sequences having a sequence complementary to one of themutant ROS polypeptides of the invention. Such isolated molecules,particularly DNA molecules, are useful as probes for gene mapping, by insitu hybridization with chromosomes, and for detecting expression of theFIG-ROS fusion protein or truncated ROS kinase polypeptide in humantissue, for instance, by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedFIG-ROS polynucleotide or truncated ROS polynucleotide of the inventionis intended fragments at least about 15 nucleotides, or at least about20 nucleotides, still more preferably at least about 30 nucleotides, orat least about 40 nucleotides in length, which are useful as diagnosticprobes and primers as discussed herein. Of course, larger fragments ofabout 50-1500 nucleotides in length are also useful according to thepresent invention, as are fragments corresponding to most, if not all,of the FIG-ROS nucleotide sequence of the cDNAs having sequences setforth in SEQ ID NOs: 1, 3, or 16. By “a fragment at least 20 nucleotidesin length”, for example, is meant fragments that include 20 or morecontiguous bases from the respective nucleotide sequences from which thefragments are derived.

Generation of such DNA fragments is routine to the skilled artisan, andmay be accomplished, by way of example, by restriction endonucleasecleavage or shearing by sonication of DNA obtainable from the cDNA clonedescribed herein or synthesized according to the sequence disclosedherein. Alternatively, such fragments can be directly generatedsynthetically.

In another aspect, the invention provides an isolated polynucleotide(e.g., a nucleotide probe) that hybridizes under stringent conditions toa mutant ROS kinase polynucleotide of the invention, such as a FIG-ROSfusion polynucleotide). The term “stringent conditions” with respect tonucleotide sequence or nucleotide probe hybridization conditions is the“stringency” that occurs within a range from about T_(m) minus 5° C.(i.e., 5° C. below the melting temperature (T_(m)) of the probe orsequence) to about 20° C. to 25° C. below T_(m). Typical stringentconditions are: overnight incubation at 42° C. in a solution comprising:50% formamide, 5×.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mMsodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate,and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. As will be understood bythose of skill in the art, the stringency of hybridization may bealtered in order to identify or detect identical or relatedpolynucleotide sequences.

By a polynucleotide or nucleotide probe that hybridizes to a referencepolynucleotide (e.g., a FIG-ROS(S) fusion polynucleotide) is intendedthat the polynucleotide or nucleotide probe (e.g., DNA, RNA, or aDNA-RNA hybrid) hybridizes along the entire length of the referencepolynucleotide or hybridizes to a portion of the referencepolynucleotide that is at least about 15 nucleotides (nt), or to atleast about 20 nt, or to at least about 30 nt, or to about 30-70 nt ofthe reference polynucleotide. These nucleotide probes of the inventionare useful as diagnostic probes and primers (e.g. for PCR) as discussedherein.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g. the FIG-ROS(S) fusion polynucleotidehaving the sequence set forth in SEQ ID NO: 3, for instance, a portion50-750 nt in length, or even to the entire length of the referencepolynucleotide, are useful as probes according to the present invention,as are polynucleotides corresponding to most, if not all, of thenucleotide sequence of the cDNAs described herein or the nucleotidesequences set forth in SEQ ID NOs: 1 or 3.

As used herein, by “a portion of a polynucleotide of ‘at least 15nucleotides’ in length”, for example, is intended 15 or more contiguousnucleotides from the nucleotide sequence of the referencepolynucleotide. As indicated, such portions are useful as nucleotideprobes for use diagnostically according to conventional DNAhybridization techniques or for use as primers for amplification of atarget sequence by the polymerase chain reaction (PCR), as described,for instance, in MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition,Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), the entire disclosureof which is hereby incorporated herein by reference. Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3′ terminal poly(A) tract of the FIG-ROS sequences (e.g., SEQ ID NOs: 1or 3) or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to aportion of a nucleic acid of the invention, since such a polynucleotidewould hybridize to any nucleic acid molecule containing a poly (A)stretch or the complement thereof (e.g., practically any double-strandedcDNA clone).

As indicated, nucleic acid molecules of the present invention, whichencode a mutant ROS kinase polypeptide of the invention, may include butare not limited to those encoding the amino acid sequence of the maturepolypeptide, by itself; the coding sequence for the mature polypeptideand additional sequences, such as those encoding the leader or secretorysequence, such as a pre-, or pro- or pre-pro-protein sequence; thecoding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain embodiments of thisaspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984). As discussed below, other such fusion proteinsinclude the FIG-ROS fusion polypeptide itself fused to Fc at the N- orC-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of a FIG-ROS fusion polypeptide or truncated ROS kinasepolypeptide disclosed herein. Variants may occur naturally, such as anatural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. See, e.g. GENES II, Lewin, B., ed., JohnWiley & Sons, New York (1985). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Some alterations included in the invention aresilent substitutions, additions and deletions, which do not alter theproperties and activities (e.g. kinase activity) of the FIG-ROS fusionpolypeptides disclosed herein.

Further embodiments of the invention include isolated polynucleotidescomprising a nucleotide sequence at least 90% identical. In someembodiments of the invention the nucleotide is at least 95%, 96%, 97%,98% or 99% identical, to a mutant ROS polynucleotide of the invention(for example, a nucleotide sequence encoding the FIG-ROS(S) fusionpolypeptide having the complete amino acid sequence set forth in SEQ IDNOs: 4, or a nucleotide sequence encoding the N-terminal of FIG and thekinase domain of ROS; or a nucleotide complementary to such exemplarysequences.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a mutant ROSpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the mutantROS polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences set forth in SEQ ID NOs: 1 and 3 or to thenucleotide sequence of the cDNA clones described herein can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711. Bestfit uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981),to find the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referenceFIG-ROS fusion polynucleotide sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referencenucleotide sequence and that gaps in homology of up to 5% of the totalnumber of nucleotides in the reference sequence are allowed.

The present invention includes in its scope nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences set forth in SEQ ID NOs: 1 or 3, or nucleotides encoding theamino acid sequences set forth in SEQ ID NOs 2, 4, D, or E, irrespectiveof whether they encode a polypeptide having ROS kinase activity. This isbecause even where a particular nucleic acid molecule does not encode afusion polypeptide having ROS kinase activity, one of skill in the artwould still know how to use the nucleic acid molecule, for instance, asa hybridization probe or a polymerase chain reaction (PCR) primer. Usesof the nucleic acid molecules of the present invention that do notencode a polypeptide having kinase include, inter alia, (1) isolatingthe FIG-ROS translocation gene or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theFIG-ROS translocation gene, as described in Verma et al., HUMANCHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York(1988); and Northern Blot analysis for detecting FIG-ROS fusion proteinmRNA expression in specific tissues.

Within the invention are also nucleic acid molecules having sequences atleast 95% identical to a nucleic acid sequence that encodes a FIG-ROSfusion polypeptide (e.g., FIG-ROS(S)) or truncated ROS lacking anextracellular domain of wild-type ROS kinase or lacking both theextracellular domain and transmembrane domain of wild-type ROS kinase.In some embodiments, the encoded Fig-ROS fusion polypeptide and/ortruncated ROS has kinase activity. Such activity may be similar, but notnecessarily identical, to the activity of the FIG-ROS fusion proteindisclosed herein (either the full-length protein, the mature protein, ora protein fragment that retains kinase activity), as measured in aparticular biological assay. For example, the kinase activity of ROS canbe examined by determining its ability to phosphorylate one or moretyrosine containing peptide substrates, for example, “Src-relatedpeptide” (RRLIEDAEYAARG), which is a substrate for many receptor andnonreceptor tyrosine kinases.

Due to the degeneracy of the genetic code, one of ordinary skill in theart will immediately recognize that a large number of the nucleic acidmolecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequence of the cDNAs described herein, tothe nucleic acid sequences set forth in SEQ ID NOs 1, 3, or 16 or tonucleic acid sequences encoding the amino acid sequences set forth inSEQ ID NOs: 2, 4, or 17 will encode a fusion polypeptide having ROSkinase activity. In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode a polypeptide that retains ROS kinase activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly effectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid). For example, guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie etal., “Deciphering the Message in Protein Sequences: Tolerance to AminoAcid Substitutions,” Science 247: 1306-1310 (1990), which describes twomain approaches for studying the tolerance of an amino acid sequence tochange.

Skilled artisans familiar with such techniques also appreciate whichamino acid changes are likely to be permissive at a certain position ofthe protein. For example, most buried amino acid residues requirenonpolar side chains, whereas few features of surface side chains aregenerally conserved. Other such phenotypically silent substitutions aredescribed in Bowie et al., supra., and the references cited therein.

Methods for DNA sequencing that are well known and generally availablein the art may be used to practice any polynucleotide embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Invitrogen), thermostable T7 polymerase (Amersham, Chicago,Ill.), or combinations of recombinant polymerases and proofreadingexonucleases such as the ELONGASE Amplification System marketed by GibcoBRL (Gaithersburg, Md.). The process may be automated with machines suchas the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier ThermalCycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNAsequencers (Applied Biosystems).

Polynucleotide sequences encoding a mutant ROS polypeptide of theinvention may be extended utilizing a partial nucleotide sequence andemploying various methods known in the art to detect upstream sequencessuch as promoters and regulatory elements. For example, one method thatmay be employed, “restriction-site” PCR, uses universal primers toretrieve unknown sequence adjacent to a known locus (Sarkar, G., PCRMethods Applic. 2: 318-322 (1993)). In particular, genomic DNA is firstamplified in the presence of primer to linker sequence and a primerspecific to the known region. Exemplary primers are those described inExample 4 herein. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16: 8186 (1988)). The primers may be designed using OLIGO 4.06Primer Analysis software (National Biosciences Inc., Plymouth, Minn.),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic.1: 111-119 (1991)). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR. Another method which may be used to retrieveunknown sequences is that described in Parker et al., Nucleic Acids Res.19: 3055-3060 (1991)). Additionally, one may use PCR, nested primers,and PROMOTERFINDER® libraries to walk in genomic DNA (Clontech, PaloAlto, Calif.). This process avoids the need to screen libraries and isuseful in finding intron/exon junctions.

When screening for full-length cDNAs, libraries that have beensize-selected to include larger cDNAs may be used or random-primedlibraries, which contain more sequences that contain the 5′ regions ofgenes. A randomly primed library is useful for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

Capillary electrophoresis systems, which are commercially available, maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) that are laser activated, anddetection of the emitted wavelengths by a charge coupled device camera.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER™ and SEQUENCE NAVIGATOR™, AppliedBiosystems) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is useful for the sequencing of small piecesof DNA that might be present in limited amounts in a particular sample.

The present invention also provides recombinant vectors that comprise anisolated polynucleotide of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof recombinant FIG-ROS polypeptides or fragments thereof by recombinanttechniques.

Recombinant constructs may be introduced into host cells usingwell-known techniques such infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells. The invention may be practiced with vectorscomprising cis-acting control regions to the polynucleotide of interest.Appropriate trans-acting factors may be supplied by the host, suppliedby a complementing vector or supplied by the vector itself uponintroduction into the host. In certain embodiments in this regard, thevectors provide for specific expression, which may be inducible and/orcell type-specific (e.g., those inducible by environmental factors thatare easy to manipulate, such as temperature and nutrient additives).

The DNA insert comprising a FIG-ROS polynucleotide or truncated ROSpolynucleotide of the invention should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promotersare known to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the mature transcripts expressed by the constructs mayinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors may include at least one selectablemarker. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracycline or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Non-limiting bacterial promoters suitable for use in the presentinvention include the E. coli lad and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (1989)CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., and Grant et al., Methods Enzymol. 153: 516-544 (1997).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY (1986).

Transcription of DNA encoding a FIG-ROS fusion polypeptide of thepresent invention by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at basepairs 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein (e.g., a GST-fusion), and may include not only secretionsignals, but also additional heterologous functional regions. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties may beadded to the polypeptide to facilitate purification. Such regions may beremoved prior to final preparation of the polypeptide. The addition ofpeptide moieties to polypeptides to engender secretion or excretion, toimprove stability and to facilitate purification, among others, arefamiliar and routine techniques in the art.

In one non-limiting example, a FIG-ROS fusion polypeptide of theinvention may comprise a heterologous region from an immunoglobulin thatis useful to solubilize proteins. For example, EP-A-0 464 533 (Canadiancounterpart 2045869) discloses fusion proteins comprising variousportions of constant region of immunoglobin molecules together withanother human protein or part thereof. In many cases, the Fc part in afusion protein is thoroughly advantageous for use in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified in the advantageous mannerdescribed. This is the case when Fc portion proves to be a hindrance touse in therapy and diagnosis, for example when the fusion protein is tobe used as antigen for immunizations. In drug discovery, for example,human proteins, such as, hIL5-has been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5 See Bennett et al., Journal of Molecular Recognition 8: 52-58(1995) and Johanson et al., The Journal of Biological Chemistry 270(16):9459-9471 (1995).

FIG-ROS polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. In some embodiments, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

Accordingly, in one embodiment, the invention provides a method forproducing a recombinant FIG-ROS fusion polypeptide by culturing arecombinant host cell (as described above) under conditions suitable forthe expression of the fusion polypeptide and recovering the polypeptide.Culture conditions suitable for the growth of host cells and theexpression of recombinant polypeptides from such cells are well known tothose of skill in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel F M et al., eds., Volume 2, Chapter 16, WileyInterscience.

In a further aspect, the invention provides a binding agent thatspecifically binds to a FIG-ROS fusion polypeptide. In some embodiments,the binding agent specifically binds to a fusion junction between a FIGportion and a ROS portion in said FIG-ROS fusion polypeptide. In someembodiments, the FIG-ROS fusion polypeptide is a FIG-ROS(S) fusionpolypeptide, a FIG-ROS(L) fusion polypeptide, or a FIG-ROS (XL) fusionpolypeptide.

In some embodiments, the binding agent of the invention is attached to adetectable label. By “detectable label” with respect to a polypeptide,polynucleotide, or binding agent disclosed herein means a chemical,biological, or other modification of or to the polypeptide,polynucleotide, or binding agent, including but not limited tofluorescence, mass, residue, dye, radioisotope, label, or tagmodifications, etc., by which the presence of the molecule of interestmay be detected. The detectable label may be attached to thepolypeptide, polynucleotide, or binding agent by a covalent ornon-covalent chemical bond.

The invention provides binding agents, such as antibodies or AQUApeptides, or binding fractions thereof, that specifically bind to theFIG-ROS fusion polypeptides (e.g., FIG-ROS(S), FIG-ROS(L), orFIG-ROS(XL) of the invention). By “specifically binding” or“specifically binds” means that a binding agent of the invention (e.g.,an antibody or AQUA peptide) interacts with its target molecule (e.g., aFIG-ROS fusion polypeptide), where the interaction is interaction isdependent upon the presence of a particular structure (i.e., theantigenic determinant or epitope) on the protein; in other words, thereagent is recognizing and binding to a specific protein structurerather than to all proteins in general. By “binding fragment thereof”means a fragment or portion of a binding reagent that specifically bindsthe target molecule (e.g., an Fab fragment of an antibody). A bindingagent that specifically binds to the target molecule may be referred toas a target specific binding agent. For example, an antibody thatspecifically binds to a FIG-ROS(L) polypeptide may be referred to as aFIG-ROS(L) specific antibody. In some embodiments, a binding agent ofthe invention has a binding affinity (K_(D)) for its target molecule(e.g., a FIG-ROS fusion polypeptide) of 1×10⁻⁶M or less. In someembodiments, a binding agent of the invention binds to its targetmolecule with a K_(D) of 1×10⁻⁷ M or less, or a K_(D) of 1×10⁻⁸ M orless, or a K_(D) of 1×10⁻⁹ M or less, or a K_(D) of 1×10⁻¹° M or less,of a K_(D) of 1×10⁻¹¹M or less, of a K_(D) of 1×10⁻¹²M or less. Incertain embodiments, the K_(D) of a binding agent of the invention forits target molecule is 1 pM to 500 pM, or between 500 pM to 1 μM, orbetween 1 μM to 100 nM, or between 100 mM to 10 nM. Non-limitingexamples of a target molecule to which a binding agent of the inventionspecifically binds to include the FIG-ROS(L) fusion polypeptide, theFIG-ROS(S) fusion polypeptide, and fragments thereof, particularly thosefragments that include the junction between the FIG portion and the ROSportion of a FIG-ROS fusion polypeptide of the invention.

The binding agent of the invention, including those useful in thepractice of the disclosed methods, include, among others, FIG-ROS fusionpolypeptide specific antibodies and AQUA peptides (heavy-isotope labeledpeptides) corresponding to, and suitable for detection andquantification of, FIG-ROS fusion polypeptide expression in a biologicalsample. Thus, a “FIG-ROS fusion polypeptide-specific binding agent” isany reagent, biological or chemical, capable of specifically binding to,detecting and/or quantifying the presence/level of expressed FIG-ROSfusion polypeptide in a biological sample. The term includes, but is notlimited to, the antibodies and AQUA peptide reagents discussed below,and equivalent binding agent are within the scope of the presentinvention.

In some embodiments, the binding agent that specifically binds to aFIG-ROS fusion polypeptide is an antibody (i.e., a FIG-ROS fusionpolypeptide-specific antibody). In some embodiments, a FIG-ROS fusionpolypeptide-specific antibody of the invention is an isolated antibodyor antibodies that specifically bind(s) a FIG-ROS fusion polypeptide ofthe invention (e.g., FIG-ROS(L), FIG-ROS (XL) or FIG-ROS(S)) but doesnot substantially bind either wild-type FIG or wild-type ROS. Alsouseful in practicing the methods of the invention are other reagentssuch as epitope-specific antibodies that specifically bind to an epitopein the extracelluar or kinase domains of wild-type ROS protein sequence(which domains are not present in the truncated ROS kinase disclosedherein), and are therefore capable of detecting the presence (orabsence) of wild type ROS in a sample.

Human FIG-ROS fusion polypeptide-specific antibodies may also bind tohighly homologous and equivalent epitopic peptide sequences in othermammalian species, for example murine or rabbit, and vice versa.Antibodies useful in practicing the methods of the invention include (a)monoclonal antibodies, (b) purified polyclonal antibodies thatspecifically bind to the target polypeptide (e.g., the fusion junctionof FIG-ROS fusion polypeptide, (c) antibodies as described in (a)-(b)above that bind equivalent and highly homologous epitopes orphosphorylation sites in other non-human species (e.g., mouse, rat), and(d) fragments of (a)-(c) above that bind to the antigen (or morepreferably the epitope) bound by the exemplary antibodies disclosedherein.

The term “antibody” or “antibodies” refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE, includingbinding fragments thereof (i.e., fragments of an antibody that arecapable of specifically binding to the antibody's target molecule, suchas F_(ab), and F(ab′)₂ fragments), as well as recombinant, humanized,polyclonal, and monoclonal antibodies and/or binding fragments thereof.Antibodies of the invention can be derived from any species of animal,such as from a mammal. Non-limiting exemplary natural antibodies includeantibodies derived from human, chicken, goats, and rodents (e.g., rats,mice, hamsters and rabbits), including transgenic rodents geneticallyengineered to produce human antibodies (see, e.g., Lonberg et al.,WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al.,WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated byreference in their entirety). Antibodies of the invention may be also bechimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26:403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851(1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may berecombinant monoclonal antibodies produced according to the methodsdisclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No.4,816,567 (Cabilly et al.) The antibodies may also be chemicallyconstructed specific antibodies made according to the method disclosedin U.S. Pat. No. 4,676,980 (Segel et al.).

Natural antibodies are the antibodies produced by a host animal, howeverthe invention contemplates also genetically altered antibodies whereinthe amino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques to thisapplication, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics. The term“humanized antibody”, as used herein, refers to antibody molecules inwhich amino acids have been replaced in the non-antigen binding regionsin order to more closely resemble a human antibody, while stillretaining the original binding ability. Other antibodies specificallycontemplated are oligoclonal antibodies. As used herein, the phrase“oligoclonal antibodies” refers to a predetermined mixture of distinctmonoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat.Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodiesconsisting of a predetermined mixture of antibodies against one or moreepitopes are generated in a single cell. In other embodiments,oligoclonal antibodies comprise a plurality of heavy chains capable ofpairing with a common light chain to generate antibodies with multiplespecificities (e.g., PCT publication WO 04/009618). Oligoclonalantibodies are particularly useful when it is desired to target multipleepitopes on a single target molecule. In view of the assays and epitopesdisclosed herein, those skilled in the art can generate or selectantibodies or mixtures of antibodies that are applicable for an intendedpurpose and desired need.

Recombinant antibodies are also included in the present invention. Theserecombinant antibodies have the same amino acid sequence as the naturalantibodies or have altered amino acid sequences of the naturalantibodies. They can be made in any expression systems including bothprokaryotic and eukaryotic expression systems or using phage displaymethods (see, e.g., Dower et al., WO91/17271 and McCafferty et al.,WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated byreference in their entirety). Antibodies can be engineered in numerousways. They can be made as single-chain antibodies (including smallmodular immunopharmaceuticals or SMIPs™), Fab and F(ab′)₂ fragments,etc. Antibodies can be humanized, chimerized, deimmunized, or fullyhuman. Numerous publications set forth the many types of antibodies andthe methods of engineering such antibodies. For example, see U.S. Pat.Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332;5,225,539; 6,103,889; and 5,260,203. The genetically altered antibodiesof the invention may be functionally equivalent to the above-mentionednatural antibodies. In certain embodiments, modified antibodies of theinvention provide improved stability or/and therapeutic efficacy.Non-limiting examples of modified antibodies include those withconservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids that do not significantlydeleteriously alter the antigen binding utility. Substitutions can rangefrom changing or modifying one or more amino acid residues to completeredesign of a region as long as the therapeutic utility is maintained.Antibodies of the invention can be modified post-translationally (e.g.,acetylation, and/or phosphorylation) or can be modified synthetically(e.g., the attachment of a labeling group). Antibodies with engineeredor variant constant or Fc regions can be useful in modulating effectorfunctions, such as, for example, antigen-dependent cytotoxicity (ADCC)and complement-dependent cytotoxicity (CDC). Such antibodies withengineered or variant constant or Fc regions may be useful in instanceswhere a parent singling protein (Table 1) is expressed in normal tissue;variant antibodies without effector function in these instances mayelicit the desired therapeutic response while not damaging normaltissue. Accordingly, certain aspects and methods of the presentdisclosure relate to antibodies with altered effector functions thatcomprise one or more amino acid substitutions, insertions, and/ordeletions. The term “biologically active” refers to a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic FIG-ROS fusionpolypeptide or truncated ROS polypeptide, or any oligopeptide thereof,to induce a specific immune response in appropriate animals or cells andto bind with specific antibodies.

Also within the invention are antibody molecules with fewer than 4chains, including single chain antibodies, Camelid antibodies and thelike and components of an antibody, including a heavy chain or a lightchain. In some embodiments an immunoglobulin chain may comprise in orderfrom 5′ to 3′, a variable region and a constant region. The variableregion may comprise three complementarity determining regions (CDRs),with interspersed framework (FR) regions for a structure FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4. Also within the invention are heavy or lightchain variable regions, framework regions and CDRs. An antibody of theinvention may comprise a heavy chain constant region that comprises someor all of a CH1 region, hinge, CH2 and CH3 region.

One non-limiting epitopic site of a FIG-ROS fusion polypeptide specificantibody of the invention is a peptide fragment consisting essentiallyof about 11 to 17 amino acids of a human FIG-ROS fusion polypeptidesequence, which fragment encompasses the fusion junction between the FIGportion of the molecule and the ROS portion of the molecule. It will beappreciated that antibodies that specifically binding shorter or longerpeptides/epitopes encompassing the fusion junction of a FIG-ROS fusionpolypeptide are within the scope of the present invention.

The invention is not limited to use of antibodies, but includesequivalent molecules, such as protein binding domains or nucleic acidaptamers, which bind, in a fusion-protein or truncated-protein specificmanner, to essentially the same epitope to which a FIG-ROS fusionpolypeptide-specific antibody or ROS truncation point epitope-specificantibody useful in the methods of the invention binds. See, e.g.,Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibodyreagents may be suitably employed in the methods of the inventionfurther described below.

Polyclonal antibodies useful in practicing the methods of the inventionmay be produced according to standard techniques by immunizing asuitable animal (e.g., rabbit, goat, etc.) with an antigen encompassinga desired fusion-protein specific epitope (e.g. the fusion junctionbetween FIG and ROS in the FIG-ROS fusion polypeptide), collectingimmune serum from the animal, and separating the polyclonal antibodiesfrom the immune serum, and purifying polyclonal antibodies having thedesired specificity, in accordance with known procedures. The antigenmay be a synthetic peptide antigen comprising the desired epitopicsequence, selected and constructed in accordance with well-knowntechniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p.75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988);Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am.Chem. Soc. 85: 21-49 (1962)). Polyclonal antibodies produced asdescribed herein may be screened and isolated as further describedbelow.

Monoclonal antibodies may also be beneficially employed in the methodsof the invention, and may be produced in hybridoma cell lines accordingto the well-known technique of Kohler and Milstein. Nature 265: 495-97(1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989).Monoclonal antibodies so produced are highly specific, and improve theselectivity and specificity of assay methods provided by the invention.For example, a solution containing the appropriate antigen (e.g. asynthetic peptide comprising the fusion junction of FIG-ROS fusionpolypeptide) may be injected into a mouse and, after a sufficient time(in keeping with conventional techniques), the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells, typically in the presence of polyethyleneglycol, to produce hybridoma cells. Rabbit fusion hybridomas, forexample, may be produced as described in U.S. Pat. No. 5,675,063. Thehybridoma cells are then grown in a suitable selection media, such ashypoxanthine-aminopterin-thymidine (HAT), and the supernatant screenedfor monoclonal antibodies having the desired specificity, as describedbelow. The secreted antibody may be recovered from tissue culturesupernatant by conventional methods such as precipitation, ion exchangeor affinity chromatography, or the like. Monoclonal Fab fragments mayalso be produced in Escherichia coli by recombinant techniques known tothose skilled in the art. See, e.g., W. Huse, Science 246: 1275-81(1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). Ifmonoclonal antibodies of one isotype are desired for a particularapplication, particular isotypes can be prepared directly, by selectingfrom the initial fusion, or prepared secondarily, from a parentalhybridoma secreting a monoclonal antibody of different isotype by usingthe sib selection technique to isolate class-switch variants(Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira etal., J. Immunol. Methods, 74: 307 (1984)). The antigen combining site ofthe monoclonal antibody can be cloned by PCR and single-chain antibodiesproduced as phage-displayed recombinant antibodies or soluble antibodiesin E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, HumanaPress, Sudhir Paul editor.)

Further still, U.S. Pat. No. 5,194,392, Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, this method involves detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971, Houghten et al. (1996)discloses linear C₁-C-alkyl peralkylated oligopeptides and sets andlibraries of such peptides, as well as methods for using sucholigopeptide sets and libraries for determining the sequence of aperalkylated oligopeptide that preferentially binds to an acceptormolecule of interest. Thus, non-peptide analogs of the epitope-bearingpeptides of the invention also can be made routinely by these methods.

Antibodies useful in the methods of the invention, whether polyclonal ormonoclonal, may be screened for epitope and fusion protein specificityaccording to standard techniques. See, e.g., Czernik et al., Methods inEnzymology, 201: 264-283 (1991). For example, the antibodies may bescreened against a peptide library by ELISA to ensure specificity forboth the desired antigen and, if desired, for reactivity only with aFIG-ROS fusion polypeptide of the invention and not with wild type FIGor wild type ROS. The antibodies may also be tested by Western blottingagainst cell preparations containing target protein to confirmreactivity with the only the desired target and to ensure no appreciablebinding to other fusion proteins involving ROS. The production,screening, and use of fusion protein-specific antibodies is known tothose of skill in the art, and has been described. See, e.g., U.S.Patent Publication No. 20050214301.

FIG-ROS fusion polypeptide-specific antibodies useful in the methods ofthe invention may exhibit some limited cross-reactivity with similarfusion epitopes in other fusion proteins or with the epitopes in wildtype FIG and wild type ROS that form the fusion junction. This is notunexpected as most antibodies exhibit some degree of cross-reactivity,and anti-peptide antibodies will often cross-react with epitopes havinghigh homology or identity to the immunizing peptide. See, e.g., Czernik,supra. Cross-reactivity with other fusion proteins is readilycharacterized by Western blotting alongside markers of known molecularweight. Amino acid sequences of cross-reacting proteins may be examinedto identify sites highly homologous or identical to the FIG-ROS fusionpolypeptide sequence to which the antibody binds. Undesirablecross-reactivity can be removed by negative selection using antibodypurification on peptide columns (e.g. selecting out antibodies that bindeither wild type FIG and/or wild type ROS).

FIG-ROS fusion polypeptide-specific antibodies of the invention that areuseful in practicing the methods disclosed herein are ideally specificfor human fusion polypeptide, but are not limited only to binding thehuman species, per se. The invention includes the production and use ofantibodies that also bind conserved and highly homologous or identicalepitopes in other mammalian species (e.g., mouse, rat, monkey). Highlyhomologous or identical sequences in other species can readily beidentified by standard sequence comparisons, such as using BLAST, withthe human FIG-ROS fusion polypeptide sequences disclosed herein (SEQ IDNOs: 1).

Antibodies employed in the methods of the invention may be furthercharacterized by, and validated for, use in a particular assay format,for example FC, IHC, and/or ICC. The use of FIG-ROS fusionpolypeptide-specific antibodies in such methods is further describedherein. The antibodies described herein, used alone or in thebelow-described assays, may also be advantageously conjugated tofluorescent dyes (e.g. Alexa488, phycoerythrin), or labels such asquantum dots, for use in multi-parametric analyses along with othersignal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker(cytokeratin) antibodies, as further described below.

In practicing the methods of the invention, the expression and/oractivity of wild type FIG and/or wild type ROS in a given biologicalsample may also be advantageously examined using antibodies (eitherphospho-specific or total) for these wild type proteins. For example,CSF receptor phosphorylation-site specific antibodies are commerciallyavailable (see CELL SIGNALING TECHNOLOGY, INC., Danvers, Mass., 2005/06Catalogue, #'s 3151, 3155, and 3154; and Upstate Biotechnology, 2006Catalogue, #06-457). Such antibodies may also be produced according tostandard methods, as described above. The amino acid sequences of bothhuman FIG and ROS are published, as are the sequences of these proteinsfrom other species.

Detection of wild type FIG and wild type ROS expression and/oractivation, along with FIG-ROS fusion polypeptide expression, in abiological sample (e.g. a tumor sample) can provide information onwhether the fusion protein alone is driving the tumor, or whether wildtype ROS is also activated and driving the tumor. Such information isclinically useful in assessing whether targeting the fusion protein orthe wild type protein(s), or both, or is likely to be most beneficial ininhibiting progression of the tumor, and in selecting an appropriatetherapeutic or combination thereof. Antibodies specific for the wildtype ROS kinase extracellular domain, which is not present in thetruncated ROS kinase disclosed herein, may be particularly useful fordetermining the presence/absence of the mutant ROS kinase.

It will be understood that more than one antibody may be used in thepractice of the above-described methods. For example, one or moreFIG-ROS fusion polypeptide-specific antibodies together with one or moreantibodies specific for another kinase, receptor, or kinase substratethat is suspected of being, or potentially is, activated in a cancer inwhich FIG-ROS fusion polypeptide is expressed may be simultaneouslyemployed to detect the activity of such other signaling molecules in abiological sample comprising cells from such cancer.

Those of skill in the art will appreciate that FIG-ROS fusionpolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of other molecules tocreate chimeric polypeptides. For example, an epitope-bearing fragmentof a FIG-ROS fusion polypeptide may be combined with the constant domainof immunoglobulins (IgG) to facilitate purification of the chimericpolypeptide and increase the in vivo half-life of the chimericpolypeptide (see, e.g., examples of CD4-Ig chimeric proteins in EPA394,827; Traunecker et al., Nature 331: 84-86 (1988)). Fusion proteinsthat have a disulfide-linked dimeric structure (e.g., from an IgGportion may also be more efficient in binding and neutralizing othermolecules than the monomeric FIG-ROS fusion polypeptide alone (seeFountoulakis et al., J Biochem 270: 3958-3964 (1995)).

In some embodiments, a binding agent that specifically binds to aFIG-ROS fusion polypeptide is a heavy-isotope labeled peptide (i.e., anAQUA peptide). Such an AQUA peptide may be suitable for the absolutequantification of an expressed FIG-ROS fusion polypeptide in abiological sample. As used herein, the term “heavy-isotope labeledpeptide” is used interchangeably with “AQUA peptide”. The production anduse of AQUA peptides for the absolute quantification or detection ofproteins (AQUA) in complex mixtures has been described. See WO/03016861,“Absolute Quantification of Proteins and Modified Forms Thereof byMultistage Mass Spectrometry,” Gygi et al. and also Gerber et al., Proc.Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which arehereby incorporated herein by reference, in their entirety). The term“specifically detects” with respect to such an AQUA peptide means thepeptide will only detect and quantify polypeptides and proteins thatcontain the AQUA peptide sequence and will not substantially detectpolypeptides and proteins that do not contain the AQUA peptide sequence.

The AQUA methodology employs the introduction of a known quantity of atleast one heavy-isotope labeled peptide standard (which has a uniquesignature detectable by LC-SRM chromatography) into a digestedbiological sample in order to determine, by comparison to the peptidestandard, the absolute quantity of a peptide with the same sequence andprotein modification in the biological sample. Briefly, the AQUAmethodology has two stages: peptide internal standard selection andvalidation and method development; and implementation using validatedpeptide internal standards to detect and quantify a target protein insample. The method is a powerful technique for detecting and quantifyinga given peptide/protein within a complex biological mixture, such as acell lysate, and may be employed, e.g., to quantify change in proteinphosphorylation as a result of drug treatment, or to quantifydifferences in the level of a protein in different biological states.

Generally, to develop a suitable internal standard, a particular peptide(or modified peptide) within a target protein sequence is chosen basedon its amino acid sequence and the particular protease to be used todigest. The peptide is then generated by solid-phase peptide synthesissuch that one residue is replaced with that same residue containingstable isotopes (¹³C, ¹⁵N). The result is a peptide that is chemicallyidentical to its native counterpart formed by proteolysis, but is easilydistinguishable by MS via a 7-Da mass shift. The newly synthesized AQUAinternal standard peptide is then evaluated by LC-MS/MS. This processprovides qualitative information about peptide retention byreverse-phase chromatography, ionization efficiency, and fragmentationvia collision-induced dissociation. Informative and abundant fragmentions for sets of native and internal standard peptides are chosen andthen specifically monitored in rapid succession as a function ofchromatographic retention to form a selected reaction monitoring(LC-SRM) method based on the unique profile of the peptide standard.

The second stage of the AQUA strategy is its implementation to measurethe amount of a protein or modified protein from complex mixtures. Wholecell lysates are typically fractionated by SDS-PAGE gel electrophoresis,and regions of the gel consistent with protein migration are excised.This process is followed by in-gel proteolysis in the presence of theAQUA peptides and LC-SRM analysis. (See Gerber et al., supra.) AQUApeptides are spiked in to the complex peptide mixture obtained bydigestion of the whole cell lysate with a proteolytic enzyme andsubjected to immunoaffinity purification as described above. Theretention time and fragmentation pattern of the native peptide formed bydigestion (e.g., trypsinization) is identical to that of the AQUAinternal standard peptide determined previously; thus, LC-MS/MS analysisusing an SRM experiment results in the highly specific and sensitivemeasurement of both internal standard and analyte directly fromextremely complex peptide mixtures.

Since an absolute amount of the AQUA peptide is added (e.g., 250 fmol),the ratio of the areas under the curve can be used to determine theprecise expression levels of a protein or phosphorylated form of aprotein in the original cell lysate. In addition, the internal standardis present during in-gel digestion as native peptides are formed, suchthat peptide extraction efficiency from gel pieces, absolute lossesduring sample handling (including vacuum centrifugation), andvariability during introduction into the LC-MS system do not affect thedetermined ratio of native and AQUA peptide abundances.

An AQUA peptide standard is developed for a known sequence previouslyidentified by the IAP-LC-MS/MS method within in a target protein. If thesite is modified, one AQUA peptide incorporating the modified form ofthe particular residue within the site may be developed, and a secondAQUA peptide incorporating the unmodified form of the residue developed.In this way, the two standards may be used to detect and quantify boththe modified an unmodified forms of the site in a biological sample.

Peptide internal standards may also be generated by examining theprimary amino acid sequence of a protein and determining the boundariesof peptides produced by protease cleavage.

Alternatively, a protein may actually be digested with a protease and aparticular peptide fragment produced can then sequenced. Suitableproteases include, but are not limited to, serine proteases (e.g.trypsin, hepsin), metallo proteases (e.g., PUMP1), chymotrypsin,cathepsin, pepsin, thermolysin, carboxypeptidases, etc.

A peptide sequence within a target protein is selected according to oneor more criteria to optimize the use of the peptide as an internalstandard. Preferably, the size of the peptide is selected to minimizethe chances that the peptide sequence will be repeated elsewhere inother non-target proteins. Thus, a peptide is preferably at least about6 amino acids. The size of the peptide is also optimized to maximizeionization frequency. Thus, in some embodiments, the peptide is notlonger than about 20 amino acids. In some embodiments, the peptide isbetween about 7 to 15 amino acids in length. A peptide sequence is alsoselected that is not likely to be chemically reactive during massspectrometry, thus sequences comprising cysteine, tryptophan, ormethionine are avoided.

A peptide sequence that does not include a modified region of the targetregion may be selected so that the peptide internal standard can be usedto determine the quantity of all forms of the protein. Alternatively, apeptide internal standard encompassing a modified amino acid may bedesirable to detect and quantify only the modified form of the targetprotein. Peptide standards for both modified and unmodified regions canbe used together, to determine the extent of a modification in aparticular sample (i.e. to determine what fraction of the total amountof protein is represented by the modified form). For example, peptidestandards for both the phosphorylated and unphosphorylated form of aprotein known to be phosphorylated at a particular site can be used toquantify the amount of phosphorylated form in a sample.

The peptide is labeled using one or more labeled amino acids (i.e., thelabel is an actual part of the peptide) or less preferably, labels maybe attached after synthesis according to standard methods. Preferably,the label is a mass-altering label selected based on the followingconsiderations: The mass should be unique to shift fragments massesproduced by MS analysis to regions of the spectrum with low background;the ion mass signature component is the portion of the labeling moietythat preferably exhibits a unique ion mass signature in MS analysis; thesum of the masses of the constituent atoms of the label is preferablyuniquely different than the fragments of all the possible amino acids.As a result, the labeled amino acids and peptides are readilydistinguished from unlabeled ones by the ion/mass pattern in theresulting mass spectrum. Preferably, the ion mass signature componentimparts a mass to a protein fragment that does not match the residuemass for any of the 20 natural amino acids.

The label should be robust under the fragmentation conditions of MS andnot undergo unfavorable fragmentation. Labeling chemistry should beefficient under a range of conditions, particularly denaturingconditions, and the labeled tag preferably remains soluble in the MSbuffer system of choice. The label preferably does not suppress theionization efficiency of the protein and is not chemically reactive. Thelabel may contain a mixture of two or more isotopically distinct speciesto generate a unique mass spectrometric pattern at each labeled fragmentposition. Stable isotopes, such as ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O or ³⁴S, aresome non-limiting labels. Pairs of peptide internal standards thatincorporate a different isotope label may also be prepared. Non-limitingamino acid residues into which a heavy isotope label may be incorporatedinclude leucine, proline, valine, and phenylalanine

Peptide internal standards are characterized according to theirmass-to-charge (m/z) ratio, and preferably, also according to theirretention time on a chromatographic column (e.g., an HPLC column).Internal standards that co-elute with unlabeled peptides of identicalsequence are selected as optimal internal standards. The internalstandard is then analyzed by fragmenting the peptide by any suitablemeans, for example by collision-induced dissociation (CID) using, e.g.,argon or helium as a collision gas. The fragments are then analyzed, forexample by multi-stage mass spectrometry (MS^(n)) to obtain a fragmention spectrum, to obtain a peptide fragmentation signature. Preferably,peptide fragments have significant differences in m/z ratios to enablepeaks corresponding to each fragment to be well separated, and asignature is that is unique for the target peptide is obtained. If asuitable fragment signature is not obtained at the first stage,additional stages of MS are performed until a unique signature isobtained.

Fragment ions in the MS/MS and MS³ spectra are typically highly specificfor the peptide of interest, and, in conjunction with LC methods, allowa highly selective means of detecting and quantifying a targetpeptide/protein in a complex protein mixture, such as a cell lysate,containing many thousands or tens of thousands of proteins. Anybiological sample potentially containing a target protein/peptide ofinterest may be assayed. Crude or partially purified cell extracts arepreferably employed. Generally, the sample has at least 0.01 mg ofprotein, typically a concentration of 0.1-10 mg/mL, and may be adjustedto a desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about10 femtomoles, corresponding to a target protein to bedetected/quantified is then added to a biological sample, such as a celllysate. The spiked sample is then digested with one or more protease(s)for a suitable time period to allow digestion. A separation is thenperformed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis,ion exchange chromatography, etc.) to isolate the labeled internalstandard and its corresponding target peptide from other peptides in thesample. Microcapillary LC is a one non-limiting method.

Each isolated peptide is then examined by monitoring of a selectedreaction in the MS. This involves using the prior knowledge gained bythe characterization of the peptide internal standard and then requiringthe MS to continuously monitor a specific ion in the MS/MS or MS^(n)spectrum for both the peptide of interest and the internal standard.After elution, the area under the curve (AUC) for both peptide standardand target peptide peaks are calculated. The ratio of the two areasprovides the absolute quantification that can be normalized for thenumber of cells used in the analysis and the protein's molecular weight,to provide the precise number of copies of the protein per cell. Furtherdetails of the AQUA methodology are described in Gygi et al., and Gerberet al. supra.

AQUA internal peptide standards (heavy-isotope labeled peptides) maydesirably be produced, as described above, to detect any quantify anyunique site (e.g., the fusion junction within a FIG-ROS fusionpolypeptide) within a mutant ROS polypeptide of the invention. Forexample, an AQUA phosphopeptide may be prepared that corresponds to thefusion junction sequence of FIG-ROS fusion polypeptide Peptide standardsfor may be produced for the FIG-ROS fusion junction and such standardsemployed in the AQUA methodology to detect and quantify the fusionjunction (i.e. the presence of FIG-ROS fusion polypeptide) in abiological sample.

For example, an exemplary AQUA peptide of the invention comprises theamino acid sequence AGSTLP, which corresponds to the three amino acidsimmediately flanking each side of the fusion junction in the second(short) variant of FIG-ROS fusion polypeptide (i.e., FIG-ROS(S) fusionpolypeptide). It will be appreciated that larger AQUA peptidescomprising the fusion junction sequence (and additional residuesdownstream or upstream of it) may also be constructed. Similarly, asmaller AQUA peptide comprising less than all of the residues of suchsequence (but still comprising the point of fusion junction itself) mayalternatively be constructed. Such larger or shorter AQUA peptides arewithin the scope of the present invention, and the selection andproduction of AQUA peptides may be carried out as described above (seeGygi et al., Gerber et al., supra.).

In another aspect, the invention provides a method for detecting aFIG-ROS gene translocation, the method comprising contacting abiological sample with a binding agent that specifically binds to aFIG-ROS fusion polypeptide (e.g., a FIG-ROS(S), FIG-ROS(XL) or aFIG-ROS(L) fusion polypeptide), where specific binding of the bindingagent to the biological sample indicates the presence of a FIG-ROS genetranslocation (e.g., that encodes a FIG-ROS(S), FIG-ROS(XL) orFIG-ROS(L) fusion polypeptide) in said biological sample.

In a further aspect, the invention provides a method for detecting aFIG-ROS gene translocation by contacting a biological sample with anucleotide probe that hybridizes to a FIG-ROS fusion polynucleotideunder stringent conditions, wherein hybridization of said nucleotideprobe to said biological sample indicates a FIG-ROS gene translocation(e.g., that encodes a FIG-ROS(S), FIG-ROS(XL), or FIG-ROS(L) fusionpolypeptide) in said biological sample.

In another aspect, the invention provides a method for identifying acancer that is likely to respond to a ROS inhibitor. The method includescontacting a biological sample of said cancer comprising at least onepolypeptide with a binding agent that specifically binds to either aFIG-ROS fusion polypeptide (e.g., a FIG-ROS(S), FIG-ROS(XL), orFIG-ROS(L) fusion polypeptide) or a mutant ROS polypeptide, whereinspecific binding of said binding agent to at least one polypeptide insaid biological sample identifies said cancer as a cancer that is likelyto respond to a ROS inhibitor. In some embodiments, the binding agent isan antibody or an AQUA peptide. In some embodiments, the cancer is froma patient (e.g., a cancer patient). In further embodiments, the cancermay be a liver cancer, a pancreatic cancer, a kidney cancer, atesticular cancer, or may be a duct cancer (e.g., a bile duct cancer ora pancreatic duct cancer).

As used herein, by “likely to respond” is meant that a cancer is morelikely to show growth retardation or abrogation in response to (e.g.,upon contact with or treatment by) a ROS inhibitor. In some embodiments,a cancer that is likely to respond to a ROS inhibitor is one that dies(e.g., the cancer cells apoptose) in response to the ROS inhibitor.

As described herein, certain normal cells (e.g., liver cells) do notexpress any ROS kinase (or show any ROS kinase activity) while cancerouscells of that cell type do. This may be, for example, because thecancerous cell expresses a truncated ROS polypeptide or a ROS fusionprotein (e.g., a FIG-ROS fusion polypeptide). The cancerous cell mayalso simply overexpress wild-type, full-length ROS kinase (where“overexpress” simply means that the cancerous cell expresses more ROSkinase than a non-cancerous cell of that same cell type). As mentionedabove, such overexpression of ROS is included in the term “mutant ROS”.For example, as described below, normal liver cells do not express ROSkinase (and do not show any ROS kinase activity) while cancerous livercells do. Thus, in some embodiments of the invention, the identificationof the presence of the ROS kinase (or the identification of the presenceof ROS kinase activity) in a cell type that does not normally expressROS (or show any ROS kinase activity) may be an indicator that the cellthus identified is a cancer that is likely to respond to a ROSinhibitor. This identification of the presence of ROS kinase (or ROSkinase activity) may be followed by further analysis of the ROS kinasewithin that cell (e.g., binding of a protein in the cell with a bindingagent that specifically binds to a mutant ROS polypeptide orhybridization of a nucleic acid molecule from the cell with a probe thathybridizes to a mutant ROS polynucleotide).

In yet another aspect, the invention provides another method foridentifying a cancer that is likely to respond to a ROS inhibitor. Themethod includes contacting a biological sample of said cancer comprisingat least one nucleic acid molecule with a nucleotide probe thathybridizes under stringent conditions to a either a FIG-ROS fusionpolynucleotide (e.g., a FIG-ROS(S), FIG-ROS(XL), or FIG-ROS(L) fusionpolynucleotide) or a mutant ROS polynucleotide, and whereinhybridization of said nucleotide probe to at least one nucleic acidmolecule in said biological sample identifies said cancer as a cancerthat is likely to respond to a ROS inhibitor. In some embodiments, theFIG-ROS fusion polynucleotide encodes a FIG-ROS(S) fusion polypeptide.In some embodiments, the FIG-ROS fusion polynucleotide encodes aFIG-ROS(L) fusion polypeptide. In some embodiments, the FIG-ROS fusionpolynucleotide encodes a FIG-ROS(XL) fusion polypeptide. In someembodiments, the cancer is from a patient (e.g., a cancer patient). Infurther embodiments, the cancer may be a liver cancer, a pancreaticcancer, a kidney cancer, a testicular cancer, or may be a duct cancer(e.g., a bile duct cancer or a pancreatic duct cancer).

The methods of the invention may be carried out in a variety ofdifferent assay formats known to those of skill in the art. Somenon-limiting examples of methods include immunoassays and peptide andnucleotide assays.

Immunoassays.

Immunoassays useful in the practice of the methods of the invention maybe homogenous immunoassays or heterogeneous immunoassays. In ahomogeneous assay the immunological reaction usually involves a mutantROS polypeptide-specific reagent (e.g. a FIG-ROS fusionpolypeptide-specific antibody), a labeled analyte, and the biologicalsample of interest. The signal arising from the label is modified,directly or indirectly, upon the binding of the antibody to the labeledanalyte. Both the immunological reaction and detection of the extentthereof are carried out in a homogeneous solution. Immunochemical labelsthat may be employed include free radicals, radio-isotopes, fluorescentdyes, enzymes, bacteriophages, coenzymes, and so forth. Semi-conductornanocrystal labels, or “quantum dots”, may also be advantageouslyemployed, and their preparation and use has been well described. Seegenerally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) andpatents cited therein.

In a heterogeneous assay approach, the reagents are usually thebiological sample, a mutant ROS kinase polypeptide-specific reagent(e.g., an antibody), and suitable means for producing a detectablesignal. Biological samples as further described below may be used. Theantibody is generally immobilized on a support, such as a bead, plate orslide, and contacted with the sample suspected of containing the antigenin a liquid phase. The support is then separated from the liquid phaseand either the support phase or the liquid phase is examined for adetectable signal employing means for producing such signal. The signalis related to the presence of the analyte in the biological sample.Means for producing a detectable signal include the use of radioactivelabels, fluorescent labels, enzyme labels, quantum dots, and so forth.For example, if the antigen to be detected contains a second bindingsite, an antibody which binds to that site can be conjugated to adetectable group and added to the liquid phase reaction solution beforethe separation step. The presence of the detectable group on the solidsupport indicates the presence of the antigen in the test sample.Examples of suitable immunoassays are the radioimmunoassay,immunofluorescence methods, enzyme-linked immunoassays, and the like.

Immunoassay formats and variations thereof, which may be useful forcarrying out the methods disclosed herein, are well known in the art.See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc.,Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold etal., “Methods for Modulating Ligand-Receptor Interactions and theirApplication”); U.S. Pat. No. 4,659,678 (Forrest et al., “Immunoassay ofAntigens”); U.S. Pat. No. 4,376,110 (David et al., “Immunometric AssaysUsing Monoclonal Antibodies”). Conditions suitable for the formation ofreagent-antibody complexes are well known to those of skill in the art.See id. FIG-ROS fusion polypeptide-specific monoclonal antibodies may beused in a “two-site” or “sandwich” assay, with a single hybridoma cellline serving as a source for both the labeled monoclonal antibody andthe bound monoclonal antibody. Such assays are described in U.S. Pat.No. 4,376,110. The concentration of detectable reagent should besufficient such that the binding of FIG-ROS fusion polypeptide isdetectable compared to background.

Antibodies useful in the practice of the methods disclosed herein may beconjugated to a solid support suitable for a diagnostic assay (e.g.,beads, plates, slides or wells formed from materials such as latex orpolystyrene) in accordance with known techniques, such as precipitation.Antibodies or other FIG-ROS fusion polypeptide-binding reagents maylikewise be conjugated to detectable groups such as radiolabels (e.g.,³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein) in accordancewith known techniques.

Cell-based assays, such flow cytometry (FC), immuno-histochemistry(IHC), or immunofluorescence (IF) are particularly desirable inpracticing the methods of the invention, since such assay formats areclinically-suitable, allow the detection of mutant ROS polypeptideexpression in vivo, and avoid the risk of artifact changes in activityresulting from manipulating cells obtained from, e.g. a tumor sample inorder to obtain extracts. Accordingly, in some embodiments, the methodsof the invention are implemented in a flow-cytometry (FC),immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.

Flow cytometry (FC) may be employed to determine the expression ofmutant ROS polypeptide in a mammalian tumor before, during, and aftertreatment with a drug targeted at inhibiting ROS kinase activity. Forexample, tumor cells from a fine needle aspirate may be analyzed by flowcytometry for FIG-ROS fusion polypeptide expression and/or activation,as well as for markers identifying cancer cell types, etc., if sodesired. Flow cytometry may be carried out according to standardmethods. See, e.g. Chow et al., Cytometry (Communications in ClinicalCytometry) 46: 72-78 (2001). Briefly and by way of example, thefollowing protocol for cytometric analysis may be employed: fixation ofthe cells with 2% paraformaldehyde for 10 minutes at 37° C. followed bypermeabilization in 90% methanol f0 minutes on ice. Cells may then bestained with the primary FIG-ROS fusion polypeptide-specific antibody,washed and labeled with a fluorescent-labeled secondary antibody. Thecells would then be analyzed on a flow cytometer (e.g. a Beckman CoulterFC500) according to the specific protocols of the instrument used. Suchan analysis would identify the level of expressed FIG-ROS fusionpolypeptide in the tumor. Similar analysis after treatment of the tumorwith a ROS-inhibiting therapeutic would reveal the responsiveness of aFIG-ROS fusion polypeptide-expressing tumor to the targeted inhibitor ofROS kinase.

Immunohistochemical (IHC) staining may be also employed to determine theexpression and/or activation status of mutant ROS kinase polypeptide ina mammalian cancer (e.g., a liver or pancreatic caner) before, during,and after treatment with a drug targeted at inhibiting ROS kinaseactivity. IHC may be carried out according to well-known techniques.See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & LaneEds., Cold Spring Harbor Laboratory (1988). Briefly, and by way ofexample, paraffin-embedded tissue (e.g. tumor tissue from a biopsy) isprepared for immunohistochemical staining by deparaffinizing tissuesections with xylene followed by ethanol; hydrating in water then PBS;unmasking antigen by heating slide in sodium citrate buffer; incubatingsections in hydrogen peroxide; blocking in blocking solution; incubatingslide in primary anti-FIG-ROS fusion polypeptide antibody and secondaryantibody; and finally detecting using ABC avidin/biotin method accordingto manufacturer's instructions.

Immunofluorescence (IF) assays may be also employed to determine theexpression and/or activation status of FIG-ROS fusion polypeptide in amammalian cancer before, during, and after treatment with a drugtargeted at inhibiting ROS kinase activity. IF may be carried outaccording to well-known techniques. See, e.g., J. M. polak and S. VanNoorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYALMICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37,BioScientific/Springer-Verlag. Briefly, and by way of example, patientsamples may be fixed in paraformaldehyde followed by methanol, blockedwith a blocking solution such as horse serum, incubated with the primaryantibody against FIG-ROS fusion polypeptide followed by a secondaryantibody labeled with a fluorescent dye such as Alexa 488 and analyzedwith an epifluorescent microscope.

A variety of other protocols, including enzyme-linked immunosorbentassay (ELISA), radio-immunoassay (RIA), and fluorescent-activated cellsorting (FACS), for measuring mutant ROS kinase polypeptides are knownin the art and provide a basis for diagnosing altered or abnormal levelsof FIG-ROS fusion polypeptide expression. Normal or standard values forFIG-ROS fusion polypeptide expression are established by combining bodyfluids or cell extracts taken from normal mammalian subjects, preferablyhuman, with antibody to FIG-ROS fusion polypeptide under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods, but preferably by photometricmeans.

Quantities of FIG-ROS fusion polypeptide expressed in subject, control,and disease samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

Peptide & Nucleotide Assays.

Similarly, AQUA peptides for the detection/quantification of expressedmutant ROS polypeptide in a biological sample comprising cells from atumor may be prepared and used in standard AQUA assays, as described indetail above. Accordingly, in some embodiments of the methods of theinvention, the FIG-ROS fusion polypeptide-specific reagent comprises aheavy isotope labeled phosphopeptide (AQUA peptide) corresponding to apeptide sequence comprising the fusion junction of FIG-ROS fusionpolypeptide, as described above.

FIG-ROS fusion polypeptide-specific reagents useful in practicing themethods of the invention may also be mRNA, oligonucleotide or DNA probesthat can directly hybridize to, and detect, fusion or truncatedpolypeptide expression transcripts in a biological sample. Such probesare discussed in detail herein. Briefly, and by way of example,formalin-fixed, paraffin-embedded patient samples may be probed with afluorescein-labeled RNA probe followed by washes with formamide, SSC andPBS and analysis with a fluorescent microscope.

Polynucleotides encoding mutant ROS kinase polypeptide may also be usedfor diagnostic purposes. The polynucleotides that may be used includeoligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.The polynucleotides may be used to detect and quantitate gene expressionin biopsied tissues in which expression of FIG-ROS fusion polypeptide ortruncated ROS polypeptide may be correlated with disease. The diagnosticassay may be used to distinguish between absence, presence, and excessexpression of FIG-ROS fusion polypeptide, and to monitor regulation ofFIG-ROS fusion polypeptide levels during therapeutic intervention.

In one embodiment, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding FIG-ROS fusion polypeptide or truncated ROS kinase polypeptideor closely related molecules, may be used to identify nucleic acidsequences that encode mutant ROS polypeptide. The construction and useof such probes is described herein. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the fusion junction, or a less specific region, e.g., the3′ coding region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingmutant ROS kinase polypeptide, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe mutant ROS polypeptide encoding sequences. The hybridization probesof the subject invention may be DNA or RNA and derived from thenucleotide sequences of SEQ ID NOs: 2 or SEQ ID NO: 16, most preferablyencompassing the fusion junction, or from genomic sequence includingpromoter, enhancer elements, and introns of the naturally occurring FIGand ROS polypeptides, as further described above.

A FIG-ROS fusion polynucleotide or truncated ROS polynucleotide of theinvention may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; or in dip stick,pin, ELISA or chip assays utilizing fluids or tissues from patientbiopsies to detect altered mutant ROS kinase polypeptide expression.Such qualitative or quantitative methods are well known in the art. In aparticular aspect, the nucleotide sequences encoding mutant ROSpolypeptide may be useful in assays that detect activation or inductionof various cancers, including cancers of the liver, pancreas, kidneys,and testes (as well as cancers that arise in the ducts, such as the bileduct, of these tissues). Mutant ROS polynucleotides may be labeled bystandard methods, and added to a fluid or tissue sample from a patientunder conditions suitable for the formation of hybridization complexes.After a suitable incubation period, the sample is washed and the signalis quantitated and compared with a standard value. If the amount ofsignal in the biopsied or extracted sample is significantly altered fromthat of a comparable control sample, the nucleotide sequences havehybridized with nucleotide sequences in the sample, and the presence ofaltered levels of nucleotide sequences encoding FIG-ROS fusionpolypeptide or truncated ROS kinase polypeptide in the sample indicatesthe presence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

Another aspect of the invention provides a method for diagnosing apatient as having a cancer or a suspected cancer driven by a ROS kinase.The method includes contacting a biological sample of said cancer or asuspected cancer (where the biological sample comprising at least onenucleic acid molecule) with a probe that hybridizes under stringentconditions to a nucleic acid molecule selected from the group consistingof a FIG-ROS fusion polynucleotide, a SLC34A2-ROS fusion polypeptide, aCD74-ROS fusion polypeptide, and a truncated ROS polynucleotide, andwherein hybridization of said probe to at least one nucleic acidmolecule in said biological sample identifies said patient as having acancer or a suspected cancer driven by a ROS kinase.

Yet another aspect of the invention provides a method for diagnosing apatient as having a cancer or a suspected cancer driven by a ROS kinase.The method includes contacting a biological sample of said cancer orsuspected cancer (where said biological sample comprises at least onepolypeptide) with a binding agent that specifically binds to a mutantROS polypeptide, wherein specific binding of said binding agent to atleast one polypeptide in said biological sample identifies said patientas having a cancer or a suspected cancer driven by a ROS kinase.

In order to provide a basis for the diagnosis of disease (e.g., acancer) characterized by expression of mutant ROS polypeptide (e.g., aFIG-ROS(S) fusion polypeptide), a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes FIG-ROSfusion polypeptide or truncated ROS kinase polypeptide, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

Additional diagnostic uses for FIG-ROS fusion polynucleotides andtruncated ROS polynucleotides (i.e., either lacking the sequencesencoding the extracellular domain of wild-type ROS or lacking thesequences encoding both the extracellular and transmembrane domains ofwild-type ROS) of the invention may involve the use of polymerase chainreaction (PCR), another assay format that is standard to those of skillin the art. See, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd.edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). PCRoligomers may be chemically synthesized, generated enzymatically, orproduced from a recombinant source. Oligomers will preferably consist oftwo nucleotide sequences, one with sense orientation (5′ to 3′) andanother with antisense (3′ to 5′), employed under optimized conditionsfor identification of a specific gene or condition. The same twooligomers, nested sets of oligomers, or even a degenerate pool ofoligomers may be employed under less stringent conditions for detectionand/or quantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of FIG-ROSfusion polypeptide or truncated ROS kinase polypeptide includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and standard curves onto which the experimental resultsare interpolated (Melby et al., J. Immunol. Methods, 159: 235-244(1993); Duplaa et al. Anal. Biochem. 229-236 (1993)). The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In another embodiment of the invention, the mutant ROS polynucleotidesof the invention may be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude fluorescence in-situ hybridization (FISH), FACS, or artificialchromosome constructions, such as yeast artificial chromosomes,bacterial artificial chromosomes, bacterial P1 constructions or singlechromosome cDNA libraries, as reviewed in Price, C. M., Blood Rev. 7:127-134 (1993), and Trask, B. J., Trends Genet. 7: 149-154 (1991).

In one embodiment, fluorescence in-situ hybridization (FISH) is employed(as described in Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York, N.Y. (1988)) and may be correlatedwith other physical chromosome mapping techniques and genetic map data.The FISH technique is well known (see, e.g., U.S. Pat. Nos. 5,756,696;5,447,841; 5,776,688; and 5,663,319). Examples of genetic map data canbe found in the 1994 Genome Issue of Science (265: 1981f). Correlationbetween the location of the gene encoding FIG-ROS fusion polypeptide ortruncated ROS polypeptide on a physical chromosomal map and a specificdisease, or predisposition to a specific disease, may help delimit theregion of DNA associated with that genetic disease. The nucleotidesequences of the subject invention may be used to detect differences ingene sequences between normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti et al.,Nature 336: 577-580 (1988)), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

It shall be understood that all of the methods (e.g., PCR and FISH) thatdetect mutant ROS polynucleotides (e.g., aberrantly expressed wild-typeROS, FIG-ROS fusion polynucleotides, SLC34A2-ROS fusion polynucleotides,and the CD74-ROS fusionpolynucleotide of the invention) may be combinedwith other methods that detect either mutant ROS polynucleotides ormutant ROS polypeptides. For example, detection of a FIG-ROSpolynucleotide in the genetic material of a biological sample (e.g.,FIG-ROS(S) in a circulating tumor cell) may be followed by Westernblotting analysis or immuno-histochemistry (IHC) analysis of theproteins of the sample to determine if the FIG-ROS(S) polynucleotide wasactually expressed as a FIG-ROS(S) fusion polypeptide in the biologicalsample. Such Western blotting or IHC analyses may be performed using anantibody that specifically binds to the polypeptide encoded by thedetected FIG-ROS(S) polynucleotide, or the analyses may be performedusing antibodies that specifically bind either to full length FIG (e.g.,bind to the N-terminus of the protein) or to full length ROS (e.g., bindan epitope in the kinase domain of ROS). Such assays are known in theart (see, e.g., U.S. Pat. No. 7,468,252).

In another example, the CISH technology of Dako allows chromatogenicin-situ hybridization with immuno-histochemistry on the same tissuesection. See Elliot et al., Br J Biomed Sci 2008; 65(4): 167-171, 2008for a comparison of CISH and FISH.

As used throughout the specification, the term “biological sample” isused in its broadest sense, and means any biological sample suspected ofcontaining a FIG-ROS fusion polypeptide, a FIG-ROS fusionpolynucleotide, a truncated ROS polynucleotide, a truncated ROSpolypeptide (i.e., either lacking the sequences encoding theextracellular domain of wild-type ROS or lacking the sequences encodingboth the extracellular and transmembrane domains of wild-type,full-length ROS), a truncated ROS polynucleotide, or a fragment thereof,and may comprise a cell, chromosomes isolated from a cell (e.g., aspread of metaphase chromosomes), genomic DNA (in solution or bound to asolid support such as for Southern analysis), RNA (in solution or boundto a solid support such as for northern analysis), cDNA (in solution orbound to a solid support), an extract from cells, blood, urine, marrow,or a tissue, and the like.

Biological samples useful in the practice of the methods of theinvention may be obtained from any mammal in which a cancercharacterized by the presence of a FIG-ROS fusion polypeptide is ormight be present or developing. As used herein, the phrase“characterized by” with respect to a cancer and indicated molecule(e.g., a ROS fusion or a mutant ROS) is meant a cancer in which a genetranslocation or mutation (e.g., causing overexpression of wild-typeROS) and/or an expressed polypeptide (e.g., a FIG-ROS fusionpolypeptide) is present, as compared to a cancer or a normal tissue inwhich such translocation, overexpression of wild-type ROS, and/or fusionpolypeptide are not present. The presence of such translocation,overexpression of wild-type ROS, and/or fusion polypeptide may drive(i.e., stimulate or be the causative agent of), in whole or in part, thegrowth and survival of such cancer or suspected cancer.

In one embodiment, the mammal is a human, and the human may be acandidate for a ROS-inhibiting therapeutic, for the treatment of acancer, e.g., a liver, pancreatic, kidney, or testicular cancer. Thehuman candidate may be a patient currently being treated with, orconsidered for treatment with, a ROS kinase inhibitor. In anotherembodiment, the mammal is large animal, such as a horse or cow, while inother embodiments, the mammal is a small animal, such as a dog or cat,all of which are known to develop cancers, including liver, kidney,testicular, and pancreatic cancers.

Any biological sample comprising cells (or extracts of cells) from amammalian cancer is suitable for use in the methods of the invention. Inone embodiment, the biological sample comprises cells obtained from atumor biopsy. The biopsy may be obtained, according to standard clinicaltechniques, from primary tumors occurring in an organ of a mammal, or bysecondary tumors that have metastasized in other tissues. In anotherembodiment, the biological sample comprises cells obtained from a fineneedle aspirate taken from a tumor, and techniques for obtaining suchaspirates are well known in the art (see Cristallini et al., Acta Cytol.36(3): 416-22 (1992)).

In some embodiments, the biological sample comprises circulating tumorcells. Circulating tumor cells (“CTCs”) may be purified, for example,using the kits and reagents sold under the trademarks Vita-Assays™,Vita-Cap™, and CellSearch® (commercially available from Vitatex, LLC (aJohnson and Johnson corporation). Other methods for isolating CTCs aredescribed (see, for example, PCT Publication No. WO/2002/020825,Cristofanilli et al., New Engl. J. of Med. 351 (8):781-791 (2004), andAdams et al., J. Amer. Chem. Soc. 130(27): 8633-8641 (July 2008)). In aparticular embodiment, a circulating tumor cell (“CTC”) may be isolatedand identified as having originated from the lung.

Accordingly, the invention provides a method for isolating a CTC, andthen screening the CTC one or more assay formats to identify thepresence of a mutant ROS polypeptide or polynucleotide of the invention(e.g., a FIG-ROS fusion polypeptide or polynucleotide) in the CTC. Somenon-limiting assay formats include Western blotting analysis,flow-cytometry (FC), immuno-histochemistry (IHC), immuno-fluorescence(IF), fluorescence in situ hybridization (FISH) and polymerase chainreaction (PCR). A CTC from a patient that is identified as comprising amutant ROS polypeptide or polynucleotide of the invention (e.g., aFIG-ROS fusion polypeptide or polynucleotide) may indicate that thepatient's originating cancer (e.g., a lung cancer such as a non-smallcell lung cancer) is likely to respond to a composition comprising atleast one ROS kinase-inhibiting therapeutic.

A biological sample may comprise cells (or cell extracts) from a cancerin which FIG-ROS fusion polypeptide or mutant ROS polypeptide (e.g.,lacking the extracellular and transmembrane domains) is expressed and/oractivated but wild type ROS kinase is not. Alternatively, the sample maycomprise cells from a cancer in which both a mutant ROS fusionpolypeptide and a wild type ROS kinase are expressed and/or activated,or in which wild type ROS kinase is expressed and/or active, but ROSfusion polypeptide is not.

Cellular extracts of the foregoing biological samples may be prepared,either crude or partially (or entirely) purified, in accordance withstandard techniques, and used in the methods of the invention.Alternatively, biological samples comprising whole cells may be utilizedin assay formats such as immunohistochemistry (IHC), flow cytometry(FC), and immunofluorescence (IF), as further described above. Suchwhole-cell assays are advantageous in that they minimize manipulation ofthe tumor cell sample and thus reduce the risks of altering the in vivosignaling/activation state of the cells and/or introducing artifactsignals. Whole cell assays are also advantageous because theycharacterize expression and signaling only in tumor cells, rather than amixture of tumor and normal cells.

In practicing the disclosed method for determining whether a compoundinhibits progression of a tumor characterized by a FIG-ROS translocationand/or fusion polypeptide, biological samples comprising cells frommammalian xenografts (or bone marrow transplants) may also beadvantageously employed. Non-limiting xenografts (or transplantrecipients) are small mammals, such as mice, harboring human tumors (orleukemias) that express a FIG-ROS fusion polypeptide (or a mutant ROSkinase containing the kinase domain but lacking the transmembrane andextracellular domains). Xenografts harboring human tumors are well knownin the art (see Kal, Cancer Treat Res. 72: 155-69 (1995)) and theproduction of mammalian xenografts harboring human tumors is welldescribed (see Winograd et al., In Vivo. 1(1): 1-13 (1987)). Similarlythe generation and use of bone marrow transplant models is welldescribed (see, e.g., Schwaller, et al., EMBO J. 17: 5321-333 (1998);Kelly et al., Blood 99: 310-318 (2002)).

In assessing mutant ROS polynucleotide presence or mutant ROSpolypeptide expression in a biological sample comprising cells from amammalian cancer tumor, a control sample representing a cell in whichsuch translocation and/or fusion protein do not occur may desirably beemployed for comparative purposes. Ideally, the control sample comprisescells from a subset of the particular cancer (e.g., bile duct livercancer) that is representative of the subset in which the mutation(e.g., FIG-ROS translocation) does not occur and/or the fusionpolypeptide is not expressed. Comparing the level in the control sampleversus the test biological sample thus identifies whether the mutantpolynucleotide and/or polypeptide is/are present. Alternatively, sinceFIG-ROS fusion polynucleotide and/or polypeptide may not be present inthe majority of cancers, any tissue that similarly does not expressmutant ROS polypeptide (or harbor the mutant polynucleotide) may beemployed as a control.

The methods described below will have valuable diagnostic utility forcancers characterized by mutant ROS polynucleotide and/or polypeptide,and treatment decisions pertaining to the same. For example, biologicalsamples may be obtained from a subject that has not been previouslydiagnosed as having a cancer characterized by since a FIG-ROStranslocation and/or fusion polypeptide, nor has yet undergone treatmentfor such cancer, and the method is employed to diagnostically identify atumor in such subject as belonging to a subset of tumors (e.g., a bileduct tumor) in which mutant ROS polynucleotide and/or polypeptide ispresent/expressed.

Alternatively, a biological sample may be obtained from a subject thathas been diagnosed as having a cancer characterized by the presence ofone type of kinase, such as EFGR, and has been receiving therapy, suchas EGFR inhibitor therapy (e.g., Tarceva™, Iressa™) for treatment ofsuch cancer, and the method of the invention is employed to identifywhether the subject's tumor is also characterized by a FIG-ROStranslocation and/or fusion polypeptide, and is therefore likely tofully respond to the existing therapy and/or whether alternative oradditional ROS-inhibiting therapy is desirable or warranted. The methodsof the invention may also be employed to monitor the progression orinhibition of a mutant ROS polypeptide-expressing cancer followingtreatment of a subject with a composition comprising a ROS-inhibitingtherapeutic or combination of therapeutics.

Such diagnostic assay may be carried out subsequent to or prior topreliminary evaluation or surgical surveillance procedures. Theidentification method of the invention may be advantageously employed asa diagnostic to identify patients having cancer, such as bile duct livercancer, characterized by the presence of the FIG-ROS fusion protein,which patients would be most likely to respond to therapeutics targetedat inhibiting ROS kinase activity. The ability to select such patientswould also be useful in the clinical evaluation of efficacy of futureROS-targeted therapeutics as well as in the future prescription of suchdrugs to patients.

The ability to selectively identify cancers in which a FIG-ROStranslocation and/or fusion polypeptide is/are present enables importantnew methods for accurately identifying such tumors for diagnosticpurposes, as well as obtaining information useful in determining whethersuch a tumor is likely to respond to a ROS-inhibiting therapeuticcomposition, or likely to be partially or wholly non-responsive to aninhibitor targeting a different kinase when administered as a singleagent for the treatment of the cancer.

Accordingly, in one embodiment, the invention provides a method fordetecting the presence of a mutant ROS polynucleotide and/or polypeptidein a cancer, the method comprising the steps of: (a) obtaining abiological sample from a patient having cancer; and (b) utilizing atleast one reagent that detects a mutant ROS polynucleotide orpolypeptide of the invention to determine whether a FIG-ROS fusionpolynucleotide and/or polypeptide is/are present in the biologicalsample.

In some embodiments, the cancer is a liver cancer, such as bile ductliver cancer. In some embodiments, the cancer is a pancreatic cancer, akidney cancer, or a testicular cancer. In other embodiments, thepresence of a FIG-ROS fusion polypeptide identifies a cancer that islikely to respond to a composition or therapeutic comprising at leastone ROS-inhibiting compound.

In some embodiments, the diagnostic methods of the invention areimplemented in a flow-cytometry (FC), immuno-histochemistry (IHC), orimmuno-fluorescence (IF) assay format. In another embodiment, theactivity of the FIG-ROS fusion polypeptide is detected. In otherembodiments, the diagnostic methods of the invention are implemented ina fluorescence in situ hybridization (FISH) or polymerase chain reaction(PCR) assay format.

The invention further provides a method for determining whether acompound inhibits the progression of a cancer characterized by a FIG-ROSfusion polynucleotide or polypeptide, said method comprising the step ofdetermining whether said compound inhibits the expression and/oractivity of said FIG-ROS fusion in said cancer. In one embodiment,inhibition of expression and/or activity of the FIG-ROS fusionpolypeptide is determined using at least one reagent that detects anFIG-ROS fusion polynucleotide or polypeptide of the invention. Compoundssuitable for inhibition of ROS kinase activity are discussed in moredetail herein.

Mutant ROS polynucleotide probes and polypeptide-specific reagentsuseful in the practice of the methods of the invention are described infurther detail above. In one embodiment, the FIG-ROS fusionpolypeptide-specific reagent comprises a fusion polypeptide-specificantibody. In another embodiment, the fusion polypeptide-specific reagentcomprises a heavy-isotope labeled phosphopeptide (AQUA peptide)corresponding to the fusion junction of FIG-ROS fusion polypeptide

The methods of the invention described above may also optionallycomprise the step of determining the level of expression or activationof other kinases, such as wild type ROS and EGFR, or other downstreamsignaling molecules in said biological sample. Profiling both FIG-ROSfusion polypeptide expression/activation and expression/activation ofother kinases and pathways in a given biological sample can providevaluable information on which kinase(s) and pathway(s) is/are drivingthe disease, and which therapeutic regime is therefore likely to be ofmost benefit.

The discovery of the mutant ROS polypeptides (e.g., the FIG-ROS fusionpolypeptides) in human cancer also enables the development of newcompounds that inhibit the activity of these mutant ROS proteins,particularly their ROS kinase activity. Accordingly, the invention alsoprovides, in part, a method for determining whether a compound inhibitsthe progression of a cancer characterized by a FIG-ROS fusionpolynucleotide and/or polypeptide, said method comprising the step ofdetermining whether said compound inhibits the expression and/oractivity of said FIG-ROS fusion polypeptide in said cancer. In oneembodiment, inhibition of expression and/or activity of the FIG-ROSfusion polypeptide is determined using at least one reagent that detectsa FIG-ROS fusion polynucleotide and/or FIG-ROS fusion polypeptide of theinvention. Non-limiting examples of such reagents of the invention havebeen described above. Compounds suitable for the inhibition of ROSkinase activity are described in more detail below.

As used herein, a “ROS inhibitor” or a “ROS-inhibiting compound” meansany composition comprising one or more compounds, chemical orbiological, which inhibits, either directly or indirectly, theexpression and/or activity of either wild type (full length) ROS or thekinase domain of ROS, either alone and/or as part of the FIG-ROS fusionpolypeptides of the invention. Such inhibition may be in vitro or invivo. “ROS inhibitor therapeutic” or “ROS-inhibiting therapeutic” meansa ROS-inhibiting compound used as a therapeutic to treat a patientharboring a cancer (e.g., a liver, testicular, kidney, or pancreaticcancer) characterized by the presence of a FIG-ROS fusion polypeptide ofthe invention.

In some embodiments of the invention, the ROS inhibitor is a bindingagent that specifically binds to a FIG-ROS fusion polypeptide, a bindingagent that specifically binds to a mutant ROS polypeptide, an siRNAtargeting a FIG-ROS fusion polynucleotide (e.g., a FIG-ROS(S) fusionpolynucleotide), or an siRNA targeting a mutant ROS polynucleotide.

The ROS-inhibiting compound may be, for example, a kinase inhibitor,such as a small molecule or antibody inhibitor. It may be a pan-kinaseinhibitor with activity against several different kinases, or akinase-specific inhibitor. Since ROS, ALK, LTK, InsR, and IGF1R belongto the same family of tyrosine kinases, they may share similar structurein the kinase domain. Thus, in some embodiments, a ROS inhibitor of theinvention also inhibits the activity of an ALK kinase an LTK kinase, aninsulin receptor, or an IGF1 receptor. ROS-inhibiting compounds arediscussed in further detail below. Patient biological samples may betaken before and after treatment with the inhibitor and then analyzed,using methods described above, for the biological effect of theinhibitor on ROS kinase activity, including the phosphorylation ofdownstream substrate protein. Such a pharmacodynamic assay may be usefulin determining the biologically active dose of the drug that may bepreferable to a maximal tolerable dose. Such information would also beuseful in submissions for drug approval by demonstrating the mechanismof drug action.

In accordance with the present invention, the FIG-ROS fusion polypeptidemay occur in at least one subgroup of human liver, pancreatic, kidney,or testicular cancer. Accordingly, the progression of a mammalian cancer(e.g., liver, pancreatic, kidney, or testicular cancer) in which FIG-ROSfusion protein is expressed may be inhibited, in vivo, by inhibiting theactivity of ROS kinase in such cancer. ROS activity in cancerscharacterized by expression of a FIG-ROS fusion polypeptide (or a mutantROS polypeptide comprising only the kinase domain) may be inhibited bycontacting the cancer (e.g., a tumor) with a ROS-inhibiting therapeutic.Accordingly, the invention provides, in part, a method for inhibitingthe progression of a FIG-ROS fusion polypeptide-expressing cancer byinhibiting the expression and/or activity of ROS kinase in the cancer.

A ROS-inhibiting therapeutic may be any composition comprising at leastone ROS inhibitor. Such compositions also include compositionscomprising only a single ROS-inhibiting compound, as well ascompositions comprising multiple therapeutics (including those againstother RTKs), which may also include a non-specific therapeutic agentlike a chemotherapeutic agent or general transcription inhibitor.

In some embodiments, a ROS-inhibiting therapeutic useful in the practiceof the methods of the invention is a targeted, small molecule inhibitor.Small molecule targeted inhibitors are a class of molecules thattypically inhibit the activity of their target enzyme by specifically,and often irreversibly, binding to the catalytic site of the enzyme,and/or binding to an ATP-binding cleft or other binding site within theenzyme that prevents the enzyme from adopting a conformation necessaryfor its activity. An exemplary small-molecule targeted kinase inhibitoris Gleevec® (Imatinib, STI-571), which inhibits CSF1R and BCR-ABL, andits properties have been well described. See Dewar et al., Blood 105(8):3127-32 (2005). Additional small molecule kinase inhibitors that maytarget ROS include TAE-684 (see examples below) and PF-02341066 (Pfizer,Inc).

PF-02341066 has the structure:

Additional small molecule inhibitors and other inhibitors (e.g.,indirect inhibitors) of ROS kinase activity may be rationally designedusing X-ray crystallographic or computer modeling of ROS threedimensional structure, or may found by high throughput screening ofcompound libraries for inhibition of key upstream regulatory enzymesand/or necessary binding molecules, which results in inhibition of ROSkinase activity. Such approaches are well known in the art, and havebeen described. ROS inhibition by such therapeutics may be confirmed,for example, by examining the ability of the compound to inhibit ROSactivity, but not other kinase activity, in a panel of kinases, and/orby examining the inhibition of ROS activity in a biological samplecomprising cancer cells (e.g., liver, pancreatic, kidney, or testicularal cancer). Methods for identifying compounds that inhibit a cancercharacterized by the expression/presence of a FIG-ROS translocationand/or fusion polypeptide, and/or mutant ROS polynucleotide and/orpolypeptide, are further described below.

ROS-inhibiting therapeutics useful in the methods of the invention mayalso be targeted antibodies that specifically bind to critical catalyticor binding sites or domains required for ROS activity, and inhibit thekinase by blocking access of ligands, substrates or secondary moleculesto a and/or preventing the enzyme from adopting a conformation necessaryfor its activity. The production, screening, and therapeutic use ofhumanized target-specific antibodies has been well-described. SeeMerluzzi et al., Adv Clin Path. 4(2): 77-85 (2000). Commercialtechnologies and systems, such as Morphosys, Inc.'s Human CombinatorialAntibody Library (HuCAL®), for the high-throughput generation andscreening of humanized target-specific inhibiting antibodies areavailable.

The production of various anti-receptor kinase targeted antibodies andtheir use to inhibit activity of the targeted receptor has beendescribed. See, e.g. U.S. Patent Publication No. 20040202655, U.S.Patent Publication No. 20040086503, U.S. Patent Publication No.20040033543, Standardized methods for producing, and using, receptortyrosine kinase activity-inhibiting antibodies are known in the art.See, e.g., European Patent No. EP1423428,

Phage display approaches may also be employed to generate ROS-specificantibody inhibitors, and protocols for bacteriophage libraryconstruction and selection of recombinant antibodies are provided in thewell-known reference text CURRENT PROTOCOLS IN IMMUNOLOGY, Colligan etal. (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section17.1. See also U.S. Pat. No. 6,319,690, U.S. Pat. No. 6,300,064, U.S.Pat. No. 5,840,479, and U.S. Patent Publication No. 20030219839.

A library of antibody fragments displayed on the surface ofbacteriophages may be produced (see, e.g. U.S. Pat. No. 6,300,064) andscreened for binding to a FIG-ROS fusion protein of the invention. Anantibody fragment that binds to a FIG-ROS fusion polypeptide isidentified as a candidate molecule for blocking constitutive activationof the FIG-ROS fusion polypeptide in a cell. See European Patent No.EP1423428.

ROS-binding targeted antibodies identified in screening of antibodylibraries as describe above may then be further screened for theirability to block the activity of ROS, both in vitro kinase assay and invivo in cell lines and/or tumors. ROS inhibition may be confirmed, forexample, by examining the ability of such antibody therapeutic toinhibit ROS kinase activity in a panel of kinases, and/or by examiningthe inhibition of ROS activity in a biological sample comprising cancercells, as described above. In some embodiments, a ROS-inhibitingcompound of the invention reduces ROS kinase activity, but reduces thekinase activity of other kinases to a lesser extent (or not at all).Methods for screening such compounds for ROS kinase inhibition arefurther described above.

ROS-inhibiting compounds that useful in the practice of the disclosedmethods may also be compounds that indirectly inhibit ROS activity byinhibiting the activity of proteins or molecules other than ROS kinaseitself. Such inhibiting therapeutics may be targeted inhibitors thatmodulate the activity of key regulatory kinases that phosphorylate orde-phosphorylate (and hence activate or deactivate) ROS itself, orinterfere with binding of ligands. As with other receptor tyrosinekinases, ROS regulates downstream signaling through a network of adaptorproteins and downstream kinases. As a result, induction of cell growthand survival by ROS activity may be inhibited by targeting theseinteracting or downstream proteins.

ROS kinase activity may also be indirectly inhibited by using a compoundthat inhibits the binding of an activating molecule necessary for ROS toadopt its active conformation. For example, the production and use ofanti-PDGF antibodies has been described. See U.S. Patent Publication No.20030219839, “Anti-PDGF Antibodies and Methods for Producing EngineeredAntibodies,” Bowdish et al. Inhibition of ligand (PDGF) binding to thereceptor directly down-regulates the receptor activity.

ROS inhibiting compounds or therapeutics may also comprise anti-senseand/or transcription inhibiting compounds that inhibit ROS kinaseactivity by blocking transcription of the gene encoding ROS and/or theFIG-ROS fusion gene. The inhibition of various receptor kinases,including VEGFR, EGFR, and IGFR, and FGFR, by antisense therapeutics forthe treatment of cancer has been described. See, e.g., U.S. Pat. Nos.6,734,017; 6,710,174, 6,617,162; 6,340,674; 5,783,683; 5,610,288.

Antisense oligonucleotides may be designed, constructed, and employed astherapeutic agents against target genes in accordance with knowntechniques. See, e.g. Cohen, J., Trends in Pharmacol. Sci. 10(11):435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988);Weintraub, H., Sci. AM pp. 40-46 (1990); Van Der Krol et al.,BioTechniques 6(10): 958-976 (1988); Skorski et al., Proc. Natl. Acad.Sci. USA (1994) 91: 4504-4508. Inhibition of human carcinoma growth invivo using an antisense RNA inhibitor of EGFR has recently beendescribed. See U.S. Patent Publication No. 20040047847. Similarly, aROS-inhibiting therapeutic comprising at least one antisenseoligonucleotide against a mammalian ROS gene or FIG-ROS fusionpolynucleotide or mutant ROS polynucleotide may be prepared according tomethods described above. Pharmaceutical compositions comprisingROS-inhibiting antisense compounds may be prepared and administered asfurther described below.

Small interfering RNA molecule (siRNA) compositions, which inhibittranslation, and hence activity, of ROS through the process of RNAinterference, may also be desirably employed in the methods of theinvention. RNA interference, and the selective silencing of targetprotein expression by introduction of exogenous small double-strandedRNA molecules comprising sequence complimentary to mRNA encoding thetarget protein, has been well described. See, e.g. U.S. PatentPublication No. 20040038921, U.S. Patent Publication No. 20020086356,and U.S. Patent Publication 20040229266.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNAse III Dicer processes dsRNA intosmall interfering RNAs (siRNA) of approximately 22 nucleotides, whichserve as guide sequences to induce target-specific mRNA cleavage by anRNA-induced silencing complex RISC (see Hammond et al., Nature (2000)404: 293-296). RNAi involves a catalytic-type reaction whereby newsiRNAs are generated through successive cleavage of longer dsRNA. Thus,unlike antisense, RNAi degrades target RNA in a non-stoichiometricmanner. When administered to a cell or organism, exogenous dsRNA hasbeen shown to direct the sequence-specific degradation of endogenousmessenger RNA (mRNA) through RNAi.

A wide variety of target-specific siRNA products, including vectors andsystems for their expression and use in mammalian cells, are nowcommercially available. See, e.g., Promega, Inc. (www.promega.com);Dharmacon, Inc. (www.dharmacon.com). Detailed technical manuals on thedesign, construction, and use of dsRNA for RNAi are available. See,e.g., Dharmacon's “RNAi Technical Reference & Application Guide”;Promega's “RNAi: A Guide to Gene Silencing.” ROS-inhibiting siRNAproducts are also commercially available, and may be suitably employedin the method of the invention. See, e.g., Dharmacon, Inc., Lafayette,Colo. (Cat Nos. M-003162-03, MU-003162-03, D-003162-07 thru -10(siGENOME™ SMARTselection and SMARTpool® siRNAs).

It has recently been established that small dsRNA less than 49nucleotides in length, and preferably 19-25 nucleotides, comprising atleast one sequence that is substantially identical to part of a targetmRNA sequence, and which dsRNA optimally has at least one overhang of1-4 nucleotides at an end, are most effective in mediating RNAi inmammals. See U.S. Patent Publication Nos. 20040038921 and 20040229266.The construction of such dsRNA, and their use in pharmaceuticalpreparations to silence expression of a target protein, in vivo, aredescribed in detail in such publications.

If the sequence of the gene to be targeted in a mammal is known, 21-23nt RNAs, for example, can be produced and tested for their ability tomediate RNAi in a mammalian cell, such as a human or other primate cell.Those 21-23 nt RNA molecules shown to mediate RNAi can be tested, ifdesired, in an appropriate animal model to further assess their in vivoeffectiveness. Target sites that are known, for example target sitesdetermined to be effective target sites based on studies with othernucleic acid molecules, for example ribozymes or antisense, or thosetargets known to be associated with a disease or condition such as thosesites containing mutations or deletions, can be used to design siRNAmolecules targeting those sites as well.

Alternatively, the sequences of effective dsRNA can be rationallydesigned/predicted screening the target mRNA of interest for targetsites, for example by using a computer folding algorithm. The targetsequence can be parsed in silico into a list of all fragments orsubsequences of a particular length, for example 23 nucleotidefragments, using a custom Perl script or commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package.

Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siRNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. See, e.g., U.S. Patent Publication No. 20030170891. An algorithmfor identifying and selecting RNAi target sites has also recently beendescribed. See U.S. Patent Publication No. 20040236517.

Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods(Graham et al. (1973) Virol. 52: 456; McCutchan et al., (1968), J. Natl.Cancer Inst. 41: 351; Chu et al. (1987), Nucl. Acids Res. 15: 1311;Fraley et al. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), Cell22: 479). DNA may also be introduced into cells using cationic liposomes(Feigner et al. (1987), Proc. Natl. Acad. Sci. USA 84: 7413).Commercially available cationic lipid formulations include Tfx 50(Promega) or Lipofectamin 200 (Life Technologies). Alternatively, viralvectors may be employed to deliver dsRNA to a cell and mediate RNAi. SeeU.S Patent Publication No. 20040023390.

Transfection and vector/expression systems for RNAi in mammalian cellsare commercially available and have been well described. See, e.g.,Dharmacon, Inc., DharmaFECT™ system; Promega, Inc., siSTRIKET™ U6Hairpin system; see also Gou et al. (2003) FEBS. 548, 113-118; Sui, G.et al. A DNA vector-based RNAi technology to suppress gene expression inmammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al.(2002) Proc. Natl. Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002)Nature Biotechnology 19, 505-508; McManus et al. (2002) RNA 8, 842-850.

siRNA interference in a mammal using prepared dsRNA molecules may thenbe effected by administering a pharmaceutical preparation comprising thedsRNA to the mammal. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene. dsRNA cantypically be administered at a dosage of less than 5 mg dsRNA perkilogram body weight per day, and is sufficient to inhibit or completelysuppress expression of the target gene. In general a suitable dose ofdsRNA will be in the range of 0.01 to 2.5 milligrams per kilogram bodyweight of the recipient per day, preferably in the range of 0.1 to 200micrograms per kilogram body weight per day, more preferably in therange of 0.1 to 100 micrograms per kilogram body weight per day, evenmore preferably in the range of 1.0 to 50 micrograms per kilogram bodyweight per day, and most preferably in the range of 1.0 to 25 microgramsper kilogram body weight per day. A pharmaceutical compositioncomprising the dsRNA is administered once daily, or in multiplesub-doses, for example, using sustained release formulations well knownin the art. The preparation and administration of such pharmaceuticalcompositions may be carried out accordingly to standard techniques, asfurther described below.

Such dsRNA may then be used to inhibit ROS expression and activity in acancer, by preparing a pharmaceutical preparation comprising atherapeutically-effective amount of such dsRNA, as described above, andadministering the preparation to a human subject having a cancer (e.g.,a liver, pancreatic, kidney, or testicular cancer) expressing FIG-ROSfusion protein or mutnat ROS polypeptide, for example, via directinjection to the tumor. The similar inhibition of other receptortyrosine kinases, such as VEGFR and EGFR using siRNA inhibitors hasrecently been described. See U.S. Patent Publication No. 20040209832,U.S. Patent Publication No. 20030170891, and U.S. Patent Publication No.20040175703.

ROS-inhibiting therapeutic compositions useful in the practice of themethods of the invention may be administered to a mammal by any meansknown in the art including, but not limited to oral or peritonealroutes, including intravenous, intramuscular, intraperitoneal,subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical(including buccal and sublingual) administration.

For oral administration, a ROS-inhibiting therapeutic will generally beprovided in the form of tablets or capsules, as a powder or granules, oras an aqueous solution or suspension. Tablets for oral use may includethe active ingredients mixed with pharmaceutically acceptable excipientssuch as inert diluents, disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloring agentsand preservatives. Suitable inert diluents include sodium and calciumcarbonate, sodium and calcium phosphate, and lactose, while corn starchand alginic acid are suitable disintegrating agents. Binding agents mayinclude starch and gelatin, while the lubricating agent, if present,will generally be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate, to delay absorption in the gastrointestinaltract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil. For intramuscular,intraperitoneal, subcutaneous and intravenous use, the pharmaceuticalcompositions of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. The carrier may consist exclusively of anaqueous buffer (“exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of theROS-inhibiting therapeutic). Such substances include, for example,micellar structures, such as liposomes or capsids, as described below.Aqueous suspensions may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

ROS-inhibiting therapeutic compositions may also include encapsulatedformulations to protect the therapeutic (e.g., a dsRNA compound or anantibody that specifically binds a FIG-ROS fusion polypeptide) againstrapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075. An encapsulatedformulation may comprise a viral coat protein. The viral coat proteinmay be derived from or associated with a virus, such as a polyoma virus,or it may be partially or entirely artificial. For example, the coatprotein may be a Virus Protein 1 and/or Virus Protein 2 of the polyomavirus, or a derivative thereof.

ROS-inhibiting compounds can also comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. For example, methods for the delivery of nucleic acidmolecules are described in Akhtar et al., 1992, Trends Cell Bio., 2,139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed.Akbtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192. U.S. Pat. No. 6,395,713 andPCT Publication No. WO 94/02595 further describe the general methods fordelivery of nucleic acid molecules. These protocols can be utilized forthe delivery of virtually any nucleic acid molecule.

ROS-inhibiting therapeutics (i.e., a ROS-inhibiting compound beingadministered as a therapeutic) can be administered to a mammalian tumorby a variety of methods known to those of skill in the art, including,but not restricted to, encapsulation in liposomes, by iontophoresis, orby incorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (see PCT Publication No. WO 00/53722).Alternatively, the therapeutic/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe composition, whether subcutaneous, intramuscular, or intradermal,can take place using standard needle and syringe methodologies, or byneedle-free technologies such as those described in Conry et al., 1999,Clin. Cancer Res., 5, 2330-2337 and PCT Publication No. WO 99/3 1262.

Pharmaceutically acceptable formulations of ROS-inhibitor therapeuticsinclude salts of the above described compounds, e.g., acid additionsalts, for example, salts of hydrochloric, hydrobromic, acetic acid, andbenzene sulfonic acid. A pharmacological composition or formulationrefers to a composition or formulation in a form suitable foradministration, e.g., systemic administration, into a cell or patient,including for example a human. Suitable forms, in part, depend upon theuse or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell. For example, pharmacological compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the composition or formulation from exerting its effect.

Administration routes that lead to systemic absorption (e.g., systemicabsorption or accumulation of drugs in the blood stream followed bydistribution throughout the entire body), are desirable and include,without limitation: intravenous, subcutaneous, intraperitoneal,inhalation, oral, intrapulmonary and intramuscular. Each of theseadministration routes exposes the ROS-inhibiting therapeutic to anaccessible diseased tissue or tumor. The rate of entry of a drug intothe circulation has been shown to be a function of molecular weight orsize. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Nonlimiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich etal, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.);and loaded nanoparticles, such as those made of polybutylcyanoacrylate,which can deliver drugs across the blood brain barrier and can alterneuronal uptake mechanisms (Prog Neuro-psychopharmacol Biol Psychiatry,23, 941-949, 1999). Other non-limiting examples of delivery strategiesfor the ROS-inhibiting compounds useful in the method of the inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

Therapeutic compositions comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes) may also be suitablyemployed in the methods of the invention. These formulations offer amethod for increasing the accumulation of drugs in target tissues. Thisclass of drug carriers resists opsonization and elimination by themononuclear phagocytic system (MPS or RES), thereby enabling longerblood circulation times and enhanced tissue exposure for theencapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes havebeen shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; PCT Publication No. WO 96/10391; PCT Publication No. WO96/10390; and PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

Therapeutic compositions may include a pharmaceutically effective amountof the desired compounds in a pharmaceutically acceptable carrier ordiluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inREMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, preservatives, stabilizers, dyes and flavoringagents can be provided. These include sodium benzoate, sorbic acid andesters of p-hydroxybenzoic acid. In addition, antioxidants andsuspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state.

The pharmaceutically effective dose depends on the type of disease, thecomposition used, the route of administration, the type of mammal beingtreated, the physical characteristics of the specific mammal underconsideration, concurrent medication, and other factors that thoseskilled in the medical arts will recognize. Generally, an amount between0.1 mg/kg and 100 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the negatively charged polymer.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient. It is understood that the specific dose level for anyparticular patient depends upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

A ROS-inhibiting therapeutic useful in the practice of the invention maycomprise a single compound as described above, or a combination ofmultiple compounds, whether in the same class of inhibitor (e.g.,antibody inhibitor), or in different classes (e.g., antibody inhibitorsand small-molecule inhibitors). Such combination of compounds mayincrease the overall therapeutic effect in inhibiting the progression ofa fusion protein-expressing cancer. For example, the therapeuticcomposition may a small molecule inhibitor, such as STI-571 (Gleevec®)alone, or in combination with other Gleevec® analogues targeting ROSactivity and/or small molecule inhibitors of EGFR, such as Tarceva™ orIressa™. The therapeutic composition may also comprise one or morenon-specific chemotherapeutic agent in addition to one or more targetedinhibitors. Such combinations have recently been shown to provide asynergistic tumor killing effect in many cancers. The effectiveness ofsuch combinations in inhibiting ROS activity and tumor growth in vivocan be assessed as described below.

The invention also provides, in part, a method for determining whether acompound inhibits the progression of a cancer (e.g., a liver,pancreatic, kidney, or testicular cancer) characterized by a FIG-ROStranslocation and/or fusion polypeptide or characterized by a mutant ROSpolynucleotide or polypeptide, by determining whether the compoundinhibits the ROS kinase activity of the mutant ROS polypeptide in thecancer. In some embodiments, inhibition of activity of ROS is determinedby examining a biological sample comprising cells from bone marrow,blood, or a tumor. In another embodiment, inhibition of activity of ROSis determined using at least one mutant ROS polynucleotide orpolypeptide-specific reagent of the invention.

The tested compound may be any type of therapeutic or composition asdescribed above. Methods for assessing the efficacy of a compound, bothin vitro and in vivo, are well established and known in the art. Forexample, a composition may be tested for ability to inhibit ROS in vitrousing a cell or cell extract in which ROS kinase is activated. A panelof compounds may be employed to test the specificity of the compound forROS (as opposed to other targets, such as EGFR or PDGFR).

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity to aprotein of interest, as described in PCT Publication No. WO 84/03564. Inthis method, as applied to FIG-ROS fusion polypeptides of the invention,large numbers of different small test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The testcompounds are reacted with the FIG-ROS fusion polypeptide, or fragmentsthereof, and washed. Bound polypeptide (e.g. FIG-ROS(L), FIG-ROS(XL), orFIG-ROS(S) fusion polypeptide) is then detected by methods well known inthe art. A purified FIG-ROS fusion polypeptide can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

A compound found to be an effective inhibitor of ROS activity in vitromay then be examined for its ability to inhibit the progression of acancer expressing FIG-ROS fusion polypeptide (such as a liver cancer,testicular cancer, kidney cancer, or a pancreatic cancer), in vivo,using, for example, mammalian xenografts harboring human liver,pancreatic, kidney, or testicular tumors (e.g., bile duct cancers) thatare express a FIG-ROS fusion polypeptide. In this procedure, cancer celllines known to express a FIG-ROS fusion protein (e.g., a FIG-ROS(S),FIG-ROS(XL), or a FIG-ROS(L)) may be placed subcutaneously in an animal(e.g., into a nude or SCID mouse, or other immune-compromised animal).The cells then grow into a tumor mass that may be visually monitored.The animal may then be treated with the drug. The effect of the drugtreatment on tumor size may be externally observed. The animal is thensacrificed and the tumor removed for analysis by IHC and Western blot.Similarly, mammalian bone marrow transplants may be prepared, bystandard methods, to examine drug response in hematological tumorsexpressing a mutant ROS kinase. In this way, the effects of the drug maybe observed in a biological setting most closely resembling a patient.The drug's ability to alter signaling in the tumor cells or surroundingstromal cells may be determined by analysis withphosphorylation-specific antibodies. The drug's effectiveness ininducing cell death or inhibition of cell proliferation may also beobserved by analysis with apoptosis specific markers such as cleavedcaspase 3 and cleaved PARP.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50. Insome embodiments, the compounds exhibit high therapeutic indices.

The following Examples are provided only to further illustrate theinvention, and are not intended to limit its scope, except as providedin the claims appended hereto. The present invention encompassesmodifications and variations of the methods taught herein which would beobvious to one of ordinary skill in the art. Materials, reagents and thelike to which reference is made are obtainable from commercial sources,unless otherwise noted.

Example 1 Identification of ROS Kinase Activity in Liver Cancer Patientsby Global Phosphopeptide Profiling

The global phosphorylation profile of kinase activation in several humanliver cancer patients, including patients XY3-78T and 090665LC, wereexamined using a recently described and powerful technique for theisolation and mass spectrometric characterization of modified peptidesfrom complex mixtures (the “IAP” technique, see U.S. Patent PublicationNo. 20030044848, Rush et al., “Immunoaffinity Isolation of ModifiedPeptides from Complex Mixtures”). The IAP technique was performed usinga phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC.,Danvers, Mass., 2003/04 Cat. #9411) to isolate, and subsequentlycharacterize, phosphotyrosine-containing peptides from extracts of livercancer cells taken from 23 human patients and para-tumor tissues.

Liver Cancer Cell Samples

Liver tumors (n=23) were collected from surgical resections frompatients when sufficient material for PhosphoScan analysis, RNA, and DNAextractions were available. According to the Edmondson grading system,all tumor samples have differentiation grades II-III. The collectedtumors were frozen in liquid nitrogen according to standard methods.

Phosphopeptide Immunoprecipitation.

A total of 0.2 g to 0.5 g tumor tissue was homogenized and lysed in urealysis buffer (20 mM HEPES pH 8.0, 9M urea, 1 mM sodium vanadate, 2.5 mMsodium pyrophosphate, 1 mM beta-glycerophosphate) at 1.25×10⁸ cells/mland sonicated. Sonicated lysates were cleared by centrifugation at20,000×g, and proteins were reduced and alkylated as describedpreviously (see Rush et al., Nat. Biotechnol. 23(1): 94-101 (2005)).Samples were diluted with 20 mM HEPES pH 8.0 to a final ureaconcentration of 2M. Trypsin (1 mg/ml in 0.001 M HCl) was added to theclarified lysate at 1:100 v/v. Samples were digested overnight at roomtemperature.

Following digestion, lysates were acidified to a final concentration of1% TFA. Phosphopeptides were prepared using the PhosphoScan kitcommercially available from Cell Signaling Technology, Inc. (Danvers,Mass.). Briefly, peptide purification was carried out using Sep-Pak C₁₈columns as described previously (see Rush et al., supra.). Followingpurification, all elutions (10%, 15%, 20%, 25%, 30%, 35% and 40%acetonitrile in 0.1% TFA) were combined and lyophilized. Dried peptideswere resuspended in 1.4 ml MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mMNa₂HPO₄, 50 mM NaCl) and insoluble material removed by centrifugation at12,000×g for 10 minutes.

The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell SignalingTechnology, Inc., Danvers, Mass.) from ascites fluid was couplednon-covalently to protein G agarose beads (Roche) at 4 mg/ml beadsovernight at 4° C. After coupling, antibody-resin was washed twice withPBS and three times with MOPS buffer. Immobilized antibody (40 μA, 160μg) was added as a 1:1 slurry in MOPS IP buffer to the solubilizedpeptide fraction, and the mixture was incubated overnight at 4° C. Theimmobilized antibody beads were washed three times with MOPS buffer andtwice with ddH₂O. Peptides were eluted twice from beads by incubationwith 40 μl of 0.1% TFA for 20 minutes each, and the fractions werecombined.

Analysis by LC-MS/MS Mass Spectrometry.

Peptides in the IP eluate (40 μl) were concentrated and separated fromeluted antibody using Stop and Go extraction tips (StageTips) (seeRappsilber et al., Anal. Chem., 75(3): 663-70 (2003)). Peptides wereeluted from the microcolumns with 1 μl of 60% MeCN, 0.1% TFA into 7.6 μlof 0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA). The samplewas loaded onto a 10 cm×75 μm PicoFrit capillary column (New Objective)packed with Magic C18 AQ reversed-phase resin (Michrom Bioresources)using a Famos autosampler with an inert sample injection valve (Dionex).The column was developed with a 45-min linear gradient of acetonitrilein 0.4% acetic acid, 0.005% HFBA delivered at 280 nl/min (Ultimate,Dionex).

Tandem mass spectra were collected as previously described (Rikova etal., Cell 131: 1190-1203-, 2007). Briefly, pTyr-containing peptides wereconcentrated on reverse-phase micro tips. LC-MS/MS analysis wasperformed with an LTQ Orbitrap Mass Spectrometer and peptide massaccuracy of 10 ppm was one of the filters used for peptideidentification (Thermo Fisher Scientific). Samples were collected withan LTQ-Orbitrap hybrid mass spectrometer, using a top-ten method, adynamic exclusion repeat count of 1, and a repeat duration of 30 sec. MSspectra were collected in the Orbitrap component of the massspectrometer and MS/MS spectra was collected in the LTQ.

Database Analysis & Assignments.

MS/MS spectra were evaluated using TurboSequest (ThermoFinnigan) (in theSequest Browser package (v. 27, rev. 12) supplied as part of BioWorks3.0). Individual MS/MS spectra were extracted from the raw data fileusing the Sequest Browser program CreateDta, with the followingsettings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20;minimum TIC, 4×10⁵; and precursor charge state, unspecified. Spectrawere extracted from the beginning of the raw data file before sampleinjection to the end of the eluting gradient. The IonQuest and VuDtaprograms were not used to further select MS/MS spectra for Sequestanalysis. MS/MS spectra were evaluated with the following TurboSequestparameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0;maximum number of differential amino acids per modification, 4; masstype parent, average; mass type fragment, average; maximum number ofinternal cleavage sites, 10; neutral losses of water and ammonia from band y ions were considered in the correlation analysis. Proteolyticenzyme was specified except for spectra collected from elastase digests.

Searches were done against the NCBI human database released on Mar. 4,2008 containing 37742 proteins allowing oxidized methionine (M+16) andphosphorylation (Y+80) as dynamic modifications.

In proteomics research, it is desirable to validate proteinidentifications based solely on the observation of a single peptide inone experimental result, in order to indicate that the protein is, infact, present in a sample. This has led to the development ofstatistical methods for validating peptide assignments, which are notyet universally accepted, and guidelines for the publication of proteinand peptide identification results (see Carr et al., Mol. Cell.Proteomics 3: 531-533 (2004)), which were followed in this Example.However, because the immunoaffinity strategy separates phosphorylatedpeptides from unphosphorylated peptides, observing just onephosphopeptide from a protein is a common result, since manyphosphorylated proteins have only one tyrosine-phosphorylated site.

For this reason, it is appropriate to use additional criteria tovalidate phosphopeptide assignments. Assignments are likely to becorrect if any of these additional criteria are met: (i) the samesequence is assigned to co-eluting ions with different charge states,since the MS/MS spectrum changes markedly with charge state; (ii) thesite is found in more than one peptide sequence context due to sequenceoverlaps from incomplete proteolysis or use of proteases other thantrypsin; (iii) the site is found in more than one peptide sequencecontext due to homologous but not identical protein isoforms; (iv) thesite is found in more than one peptide sequence context due tohomologous but not identical proteins among species; and (v) sitesvalidated by MS/MS analysis of synthetic phosphopeptides correspondingto assigned sequences, since the ion trap mass spectrometer produceshighly reproducible MS/MS spectra. The last criterion is routinelyemployed to confirm novel site assignments of particular interest.

All spectra and all sequence assignments made by Sequest were importedinto a relational database. Assigned sequences were accepted byfiltering for XCorr values of at least 1.5 and Mass Error Range within10 ppm.

The foregoing IAP analysis identified many tyrosine phosphorylatedproteins, the majority of which are novel (data not shown). Among the 23patients with liver cancer, three had bile duct liver cancer. Twopatients with bile duct liver cancer, namely patients XY3-78T and090665LC, had liver cancer samples that were found to contain tyrosinephosphorylated ROS kinase, which was not detected by MS analysis intissue adjacent to tumor nor in any of the remaining 21 patient samples.

Example 2 Isolation & Sequencing of FIG-ROS Fusion Gene

Given the presence of the activated form of ROS kinase detected in twoliver cancer patient samples, 5′ rapid amplification of cDNA ends on thesequence encoding the kinase domain of ROS was conducted in order todetermine whether a chimeric ROS transcript was present.

Rapid Amplification of Complementary DNA Ends

RNeasy Mini Kit (Qiagen) was used to extract RNA from human tumorsamples. DNA was extracted with the use of DNeasy Tissue Kit (Qiagen).Rapid amplification of cDNA ends was performed with the use of 5′ RACEsystem (Invitrogen) with primers ROS-GSP1 for cDNA synthesis andROS-GSP2 and ROS-GSP3.1 for a nested PCR reaction, followed by cloningand sequencing PCR products.

For the 5′RACE system, the following primers were used:

(SEQ ID NO: 27) ROS-GSP1: 5′ACCCTTCTCGGTTCTTCGTTTCCAFor the nested PCR reaction, the following primers were used.

(SEQ ID NO: 28) ROS-GSP2: 5′TCTGGCGAGTCCAAAGTCTCCAAT (SEQ ID NO: 29)ROS-GSP3.1: 5′CAGCAAGAGACGCAGAGTCAGTTT

Sequencing of the PCR products revealed that the ROS kinases in thepatient samples of XY3-78T and 090665LC, were indeed products of achimeric ROS transcript, namely a fusion of part of a ROS transcriptwith part of a transcript of a FIG gene. Sequence analysis revealed thatboth patients XY3-78T and 090665LC had liver cancer cells that containedfusion protein resulting from the fusion of the c-terminus of ROS to theN-terminus of FIG (see FIG. 2, panel B and C). The FIG-ROS fusions inboth samples were in-frame. In patient XY3-78T, a shorter fusionprotein, namely FIG-ROS(S) resulted from the fusion of the first 209amino acids of FIG to the last 421 amino acids of ROS. In patient090665LC, a longer fusion protein, namely FIG-ROS(L) resulted from thefusion of the first 412 amino acids of FIG to the last 466 amino acidsof ROS.

In addition, a third FIG-ROS fusion is discovered (FIG-ROS (XL), wherethe fusion occurs after exon 7 of the FIG gene and before exon 32 of theROS gene. The nucleic acid sequence for the coding region of fusion geneis provided in SEQ ID NO: 16 and the amino acid sequence for the fusionpolypeptide encoded by the fusion gene is provided in SEQ NO: 17.

Example 3 Detection of Mutant ROS Kinase Expression in a Human CancerSample Using PCR Assay

The presence of mutant ROS kinase and/or a FIG-ROS fusion protein of theinvention (e.g., FIG-ROS(S) or FIG-ROS(S)) in a human cancer sample wasdetected using cDNA or genomic reverse transcriptase (RT) and/orpolymerase chain reaction (PCR). These methods have been previouslydescribed. See, e.g., Cools et al.; N Engl. J. Med. 348: 1201-1214(2003).

PCR Assay

To confirm that the FIG-ROS fusion had occurred, RT-PCR was performed onRNA extracted from the liver cancer cell samples of patients XY3-78T and090665LC. For RT-PCR, first-strand cDNA was synthesized from 2.5 ug oftotal RNA with the use of SuperScript™ III first-strand synthesis system(Invitrogen) with oligo (dT)₂₀. Then, the FIG-ROS fusion gene wasamplified with the use of primer pairs FIG-F2 and ROS-GSP3.1. Theirsequences are:

(SEQ ID NO: 30) FIG-F2: 5′ACTGGTCAAAGTGCTGACTCTGGT (SEQ ID NO: 31)ROS-GSP3.1: 5′CAGCAAGAGACGCAGAGTCAGTTT

As shown on FIG. 3, patient XY3-78T's liver cancer cell samplescontained mRNA predicted to encode the FIG-ROS(S) fusion polypeptide.The liver cancer cell samples from patient 090665LC contained mRNApredicted to encode the FIG-ROS(L) fusion polypeptide. As a control,RT-PCR was conducted on RNA isolated from the U118MG cell line, a humanglioblastoma known to contain the FIG-ROS(S) translocation. U118 MGcells were purchased from American Type Culture Collection (Manassas,Va.) and grown in DMEM with 10% FBS.

To determined whether the liver cell samples from patient 090665LC,liver cell samples from patient XY3-78T's, or the U118MG humanglioblastoma cell line expressed full length FIG or full length ROS,RT-PCR was performed using the FIG-F2 and ROS-GSP3.1 primers to amplifythe FIG-ROS translocation, as well as the following primers pairs toamplify wild-type FIG (i.e., full-length FIG), wild-type ROS, and, as acontrol, wild-type GAPDH.

Wild type FIG gene was amplified with the use of primer pairs FIG-F3 andFIG-R8.

(SEQ ID NO: 32) FIG-F3: 5′TTGGATAAGGAACTGGCAGGAAGG (SEQ ID NO: 33)FIG-R8: 5′ACCGTCATCTAGCGGAGTTTCACT

Wild-type ROS gene was amplified using primer pairs ROS-Ex31F andROS-GSP2.

(SEQ ID NO: 34) ROS-Ex31F: 5′AGCCAAGGTCCTGCTTATGTCTGT (SEQ ID NO: 35)ROS-GSP2: 5′TCTGGCGAGTCCAAAGTCTCCAAT

Wild-type GAPDH was amplified using primer pairs GAPDH-F and GAPDH-R

(SEQ ID NO: 36) GAPDH-F: 5′TGGAAATCCCATCACCATCT (SEQ ID NO: 37)GAPDH-R: 5′GTCTTCTGGGTGGCAGTGAT

As shown in FIG. 4, liver cancer cells from patients XY3-78T and090665LC express wild-type FIG, but neither expresses wild-type ROS. TheU118MG cell line expresses neither wild-type FIG nor wild-type ROS.HCC78 a human non-small cell lung cancer cell line, which contains anSLC34A2-ROS translocation, served as a negative control. HCC78 cellswere purchased from the ATCC (Manassas, Va.), and were maintained inDMEM with 10% FBS.

For genomic PCR, DNA was extracted from the cell samples with the use ofDNeasy Tissue Kit (Qiagen). PCR amplification of the fusion gene wasperformed with the use of LongRange PCR kit (Qiagen) with primer pairsFIG-F3 and ROS-GSP3.1 for XY3-78T.

(SEQ ID NO: 38) FIG-F3: 5′ TTGGATAAGGAACTGGCAGGAAGG (SEQ ID NO: 39)ROS-GSP3.1: 5′ CAGCAAGAGACGCAGAGTCAGTTT

PCR amplification of the fusion gene was performed with the use ofLongRange PCR kit (Qiagen) with primer pairs FIG-F7 and ROS-GSP4.1 for090665LC and UI 18MG.

(SEQ ID NO: 40) FIG-F7: 5′ TGTGGCTCCTGAAGTGGATTCTGA (SEQ ID NO: 41)ROS-GSP4.1: 5′GCAGCTCAGCCAACTCTTTGTCTT

As shown in FIG. 5, the FIG-ROS translocation occurred in the genome ofthe liver cancer cells of patients XY3-78T and 090665LC. Although theU118MG cell line expresses the same FIG-ROS(L) fusion polypeptide as thecells of patient 090665LC, the exact genomic breakpoints in FIG and ROSgene between these two samples are different. The breakpoints were foundto be:

XY3-78T 1-822 bp of FIG-Intron3 659-619 bp of ROS-Intron35 660-1228 bpof ROS Intron35 090665LC 1-2402 bp of FIG-Intron7

2317-2937 bp of ros-Intron34

U118MG 1-2304 bp of FIG-Intron7

583-2937 bp ros-Intron34

The nucleotide sequence of intron 3 of the human FIG gene is providedherewith as SEQ ID NO:5. The nucleotide sequence of intron 7 of thehuman FIG gene is provided herewith as SEQ ID NO: 6. The nucleotidesequence of intron 34 of the human ROS gene is provided herewith as SEQID NO: 7. The nucleotide sequence of intron 35 of the human ROS gene isprovided herewith as SEQ ID NO: 8.

This assay may be used to detect the presence of a mutant ROS kinaseand/or a FIG-ROS fusion protein of the invention (e.g., FIG-ROS(S) orFIG-ROS(S) in a human cancer sample in other biological tissue samples(e.g., tumor tissue samples may be obtained from a patient having liver,pancreatic, kidney, or testicular cancer). Such an analysis willidentify a patient having a cancer characterized by expression of thetruncated ROS kinase (and/or FIG-ROS fusion protein), which patient islikely to respond to treatment with a ROS inhibitor.

Example 4 Generation of Recombinant Retrovirus Encoding Fig-Ros FusionPolypeptides

The open reading frame of the FIG-ROS (L) and FIG-ROS(S) fusion gene wasamplified by PCR from cDNA isolated from patients 090665LC and XY3-78T,respectively, using the following pair of primers (FIG-Fc:5′ATGTCGGCGGGCGGTCCATG (SEQ ID NO: 42); ROS-Rc:5′TTAATCAGACCCATCTCCAT(SEQ ID NO: 43)). These PCR products were cloned into the retroviralvector MSCV-Neo with a C-terminal Myc tag (EQKLISEEDL (SEQ ID NO: 44);(MSCV-neo vector and MSCV-puro vector are commercially available fromClontech.). Additional recombinant retroviral constructs (e.g., emptyMSCV-neo vector, MSCV-puro-src, etc.) were also generated. TheFIG-ROS(S) containing MSCV-Neo vector was deposited with the AmericanType Culture Collection (“ATCC”, Manassas, Va.) under the terms of theBudapest Treaty on Jan. 21, 2009 and assigned ATCC Patent DepositDesignation No. PTA-9721.

The resulting recombinant retroviral constructs (i.e., containingFIG-ROS(S) or FIG-ROS(L)) were transfected into 293T cells to bepackaged into recombinant retrovirus capable of infecting (and therebytransducing) cells. To do this, 293T cells (e.g., commercially availablefrom ATCC) were maintained in 10% DMEM containing 10% fetal bovine serumin 10 cm tissue culture plates. 24-48 hours prior to transfection, the293T cells were plated at about 50-80% confluency. Transfection wasperformed using the FuGENE reagent (commercially available from RocheDiagnostics), according to the manufacturer's instructions. Typically,for each recombinant construct, a 3:1 ratio of the FuGENE reagent (inul) to DNA (ug) was used (e.g., 3 ul FuGENE to 1 ug Myc-taggedFIG-ROS(S) in MSCV-Neo). 48 hours following transfection, the media wasremoved, and any cells within the media (now containing recombinantvirus) was removed by filtering the media through a 0.45 um syringefilter. The media (also referred to as viral soup) was stored at −80° C.

Example 5 Expression of FIG-ROS Fusion Proteins in 3T3 Cells

3T3 cells were purchased from American Type Culture Collection(Manassas, Va.). 3T3 cells were grown at 37° C. in DMEM media with 10%FBS.

1 ml of recombinant retrovirus encoding the Fig-Ros fusion polypeptidesgenerated as described in Example 4 were used to transducer 3T3 cellsfrom 10 cm plate with 50% confluency. In addition, an empty retrovirus(i.e., generated from an empty MSCV-Neo vector with a C-terminal Myc tagwas transduced into 3T3 cells as a control.

3T3 cells were infected with (i.e., transduced with) recombinantretrovirus expressing FIG-ROS(S) from XY3-78T, FIG-ROS(L) from 090665LC.Empty retrovirus was also used to infect 3T3 cells as a control. Twodays after transduction, 0.5 mg/ml G418 was added to the cell culturemedia. Two weeks after being transduced (i.e., 12 days after selectionin G418), 1 million cells were lysed and Western blotting analysisperformed, staining the electrophoretically resolved cell lysates withan antibody that specifically bound to the kinase domain of ROS, as wellas a phospho-antibody against ROS. The cell lysates were also probedwith antibodies against several downstream signaling substrates of ROSkinase including p-STAT3 (i.e., phosphorylated STAT3), STAT3, p-AKT(i.e., phosphorylated AKT), and AKT. b-actin was also stained to ensurethat equivalent amounts of lysates were present in all lanes. Allantibodies are from Cell Signaling Technology, Inc.

As shown in FIG. 6, the 3T3 cells transduced with recombinant retrovirusstably expressed FIG-ROS(S) and FIG-ROS(L). As expected, the NC (emptyvector) cells did not express any ROS. Expression of FIG-ROS(S) andFIG-ROS(L) activate downstream signaling molecules, STAT3 and AKT.

Example 6 Effect of FIG-ROS Fusion Proteins on 3T3 Cells' Growth InVitro and In Vivo

3T3 cells have contact inhibition, meaning that they do not formcolonies in soft agar. To determine if the presence of active ROS kinasein these cells removed their contact inhibition, retrovirally transduced3T3 cells were selected for G418 (0.5 mg/ml) for 7 days, and the cellswere then cultured in soft agar in triplicate for 17 days. A retrovirusencoding the short version of SLC34A2-ROS was also used to transduce 3T3cells. As a control, a retrovirus encoding the src kinase was also usedto transducer 3T3 cells. The protocol for soft agar assay is attached.

As shown in FIG. 7, 3T3 cells transduced with either src kinase- orFIG-ROS(S)-encoding retrovirus lost their contact inhibitiondramatically. This provides evidence that the presence of FIG-ROS(S) isable to drive a cell into a cancerous state of growth. The presence ofFIG-ROS(L) also enabled 3T3 cells to lose their contact inhibition (seeFIG. 7, top left panel), as did SLC34A2-ROS(S) (data not shown),although the effect was not as significant as that seen with FIG-ROS(S).

In addition, the ability of transduced 3T3 cells to form tumors in vivowas analyzed. Immunocompromised nude mice (which lack a thymus,available from the Jackson Laboratory, Bar Harbor, Me.) were injectedwith 1×10⁶ 3T3 cells transduced with retrovirus containing empty vector,FIG-ROS(L) or FIG-ROS(S). Mice were monitored daily for tumor formationand size, and were sacrificed when tumors reached approximately 1 cm×1cm.

As shown in FIG. 8, two weeks after being injected with 3T3 cellstransduced with either FIG-ROS(S) or FIG-ROS(L), tumor formation wasapparent in the injected nude mice.

Example 7 Subcellular Localization of FIG-ROS(L) and FIG-ROS(S) in 3T3Cells

Recombinant vectors were generated to expressed Myc-tagged versions ofFIG-ROS(L) and FIG-ROS(S), where the myc tag was incorporated onto theC-terminus of the FIG-ROS fusion polypeptide. 3T3 cells were stablytransfected with the recombinant expression vectors or with an empty“neo” only vector (control)

Immunoflurescence was performed with a standard protocol (publiclyavailable from Cell Signaling Technology, Inc.). Briefly, The 9E1H1D9ROS antibody, Myc-Tag antibody (CST# 2278) and the Golgin-97 antibodywere from Cell Signaling Technology, Inc. (Danvers, Mass.).

As shown in FIGS. 9A and 9B, the two different FIG-ROS fusionpolypeptides of the invention localized to different areas of the cell.FIG-ROS(L) localized to Golgi apparatus, and co-localizes with the Golgimarker (golgin-97) (see images under “Myc-FR(L)” in both FIGS. 9A and9B). To our surprise, the staining pattern of FIG-ROS(S) was cytoplasmic(see images under “Myc-FR(S)” in both FIGS. 9A and 9B), even though itcontains the second coiled-coil domain of FIG, suggesting that thecoiled-coil domain of FIG is necessary, but not sufficient to targetFIG-ROS(S) to the Golgi apparatus. This may be because the PDZ domain ofFIG is present in FIG-ROS(L), but not in FIG-ROS(S). Interestingly,SLC34A2-ROS(S) was localized to para-nuclei compartment (see imagesunder “Myc-SR(S)” in both FIGS. 9A and 9B). The fact that theSLC34A2-ROS(S) fusion, which contains transmembrane domain of ROS, islocalized in perinuclear compartment suggests that transmembrane domainof ROS also contributes to its localization.

Thus, different ROS fusions have distinct subcellular localization,suggesting that they may activate different substrates in vivo.

Example 8 FIG-ROS(L) and FIG-ROS(S) Activity in Transduced BaF3 Cells

Murine BaF3 cells normally need interleukin-3 (IL-3) to survive. BaF3cells were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Germany) and were maintained at 37° C. in RPMI-1640medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma) and 1.0ng/ml murine IL-3 (R&D Systems).

To determine if expression of a FIG-ROS fusion polypeptide of theinvention could enable BaF3 cells to survive without IL-3, we transducedBaF3 cells with the retroviruses described in Example 4 encodingFIG-ROS(L) and FIG-ROS(S). In addition, retrovirus encoding theFIG-ROS(L) from U118MG were also generated and used to transducer BaF3cells.

As shown in FIG. 10, FIG-ROS(S), FIG-ROS(L), and FIG-ROS(L) from U118MGwere stably expressed in BaF3 cells grown with or without IL-3. Indeed,as shown in FIG. 11, we found that the presence of FIG-ROS(L) orFIG-ROS(S) enabled BaF3 cells to grow in the absence of IL-3.Interestingly, FIG-ROS(S) expressing BaF3 cells grew at a faster pacethan the BaF3 expressing FIG-ROS(L).

Next, an in vitro kinase assay was performed to determine if the ROSkinase portion of the FIG-ROS fusion polypeptides was active. Celllysates from FIG-ROS transduced BaF3 cells were subjected toimmunoprecipitation with anti-Myc-Tag antibody (which pulls down theMyc-tagged FIG-ROS fusion polypeptides). The pulled-down ROS immunecomplex were washed 3 times with cell lysis buffer, followed by kinasebuffer (Cell Signaling Technology). Kinase reactions were initiated byre-suspending the ROS immune complex into 25 ul kinase buffer thatcontains 50 uM ATP, 0.2 uCi/ul [gamma32p] ATP, with 1 mg/ml of eitherPoly (EY, 4:1). Reactions were stopped by spotting reaction cocktailonto p81 filter papers. Samples were then washed and assayed for kinaseactivity by detection with a scintillation counter. As shown in FIG. 12,while both FIG-ROS (L) and FIG-ROS(S) can phosphorylate its substrate,FIG-ROS(S) is more potent than FIG-ROS(L). In other words, FIG-ROS(S)has a much higher kinase activity than FIG-ROS(L). Equal loading of thelanes is shown in the Western blotting analysis of the ROS immunecomplexes using a ROS-specific antibody (see FIG. 12, lower panel).

The higher potency of FIG-ROS(S) as compared to FIG-ROS(L) is consistentwith data from soft agar assay (see FIG. 7) and IL-3 independent growthassay (see FIG. 11).

Example 9 Sensitivity of FIG-ROS(L) and FIG-ROS(S) to TAE-684

The small molecule, TAE-684, a 5-chloro-2,4-diaminophenylpyrimidine,which has the structure:

and has been shown to inhibit the ALK kinase. Galkin, et al., Proc.National Acad. Sci. 104(1) 270-275, 2007.

In this example, we determined whether or not TAE-684 also inhibitedFIG-ROS fusion polypeptide. To do this, BaF3 and Karpas 299 cells wereobtained from DSMZ (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Germany). BaF3 cells were maintained as describedabove and Karpas 299 cells (a lymphoma cell line) were grown inRPMI-1640 with 10% FBS.

BaF3 cells were transduced with retrovirus encoding FIG-ROS(S),FIG-ROS(L), or FLT-31TD (the Internal tandem duplication mutation inFLT3 causes AML leukemia), and selected for IL3 independent growth.Karpas 299 cells, which express NPM-ALK, was used as a positive control.

A MTS assay was performed using the CellTiter 96 Aqueous One SolutionReagent, (Promega, Catalog No, G3582), Briefly, 1×10⁵ cells/well in 24well plate were grown in 1 ml medium that included 0 nM, 3 nM, 10 nM, 30nM, 100 nM, 300 nM or 1000 nM TAE-684. After 72 hours, 20 ul of theCellTiter 96 Aqueous One Solution Reagent was added into each well of a96 well assay plate (flat bottom), and then 100 ul of cells grown withor without treatment. Media-only wells were used as controls. The 96well plate was incubated for 1-4 hours at 37° C., and then viable cellswere counted by reading the absorbance at 490 nm using a 96 well platereader.

As shown in FIG. 13, the BaF3 cells transduced with retrovirusexpressing one of the FIG-ROS polypeptides stopped growing in thepresence of TAE-684. Interestingly, FIG-ROS(S) is less susceptible toTAE-684 than FIG-ROS(L). Karpas 299 cells also responded (i.e., stoppedgrowing) in the presence of TAE-684, which was expected since theyexpress ALK and TAE-684 inhibits the ALK kinase. The BaF3 cellstransduced with FLT3/ITD were not susceptible to TAE-684.

The mechanism of death of the BaF3 and Karpas 299 cells was nextreviewed by measuring the percentage of cleaved-caspase 3 positive cellsby flow cytometry assay using cleaved caspase-3 as a marker forapoptosis. These results were obtained using the protocol publicallyavailable from Cell Signaling Technology, Inc. (Danvers, Mass.)

As shown in FIG. 14, the presence of TAE-684 caused the BaF3 cellsexpressing FIG-ROS(S) or FIG-ROS(L) to die by apoptosis. Interestingly,Karpas 299 cells, which stop growing in the presence of TAE-684, did notdie by apoptosis—they simply underwent cell cycle arrest. Thus, themechanism by which TAE-684 inhibits FIG-ROS fusion polypeptides islikely different from the mechanism by which TAE-684 inhibits the ALKkinase.

To further identify the mechanism of action of TAE-684 on the FIG-ROSfusion polypeptides of the invention, all four cell lines (i.e., Karpas299 cells and BaF3 cells transduced with retrovirus encoding FIG-ROS(S),FIG-ROS(L), and FLT-31TD) were subjected to Western blotting analysisfollowing treatment with 0, 10, 50, or 100 nM TAE-684 for three hours.All antibodies were from Cell Signaling Technology, Inc.

As shown in FIG. 15, phosphorylation of both FIG-ROS(S) and FIG-ROS(L)in FIG-ROS(S) and FIG-ROS(L) expressing BaF3 cells was inhibited byTAE-684. In addition, phosphorylation of STAT3, AKT, and ERK, and Shp2were inhibited in FIG-ROS(S) and FIG-ROS(L) expressing BaF3 cells. Thephosphorylation of STAT3, AKT, and ERK, and Shp2 was not affected in theBaF3 cells transduced with the FLT-31TD retrovirus. TAE-684 alsoinhibited ALK and ERK phosphorylation in Karpas 299 cells. Since ROS,ALK, LTK, InsR, and IGF1R belong to the same family of tyrosine kinases,they may share similar structure in the kinase domain. Kinase inhibitorsor antibodies designed against ALK, LTK, InsR, and IGF1R may havetherapeutic effects against ROS kinase.

Example 10 Detection of Mutant ROS Expression in a Human Cancer SampleUsing FISH Assay

The presence of a ROS fusion polynucleotide (e.g., a FIG-ROS(L),FIG-ROS(S), FIG-ROS(XL), SLC34A2-ROS(S), SLC34A2-ROS(VS),SLC34A2-ROS(L), or CD74-ROS) in liver cancer (e.g., in acholangiocarcinoma), pancreatic cancer, kidney cancer, or testicularcancer is detected using a fluorescence in situ hybridization (FISH)assay. Such FISH assays are well known in the art (see, e.g., Verma etal. Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NewYork, N.Y. (1988).

To do this, paraffin-embedded human tumor samples are examined. Sometissues that are examined include liver, pancreas, testicular, andkidney cancers, particularly cancers affecting the ducts of all of thesetissues.

For analyzing rearrangements involving the ROS gene, a dual colorbreak-apart probe can be designed. As shown in FIG. 16, several BACprobes surround the FIG and ROS genes on chromosome 6. While theseprobes are ideal for identifying translocations between the FIG gene(also known as the GOPC gene—see FIG. 16) and the ROS gene, these probescan also be used to identify other ROS gene translocation.

For these studies, a proximal probe (BAC clone RP1-179P9) and two distalprobes (BAC clone RP11-323017, RP1-94G16) (all of which are commerciallyavailable, for example, from Invitrogen Inc., Carlsbad, Calif., asCatalog Nos. RPCI1.C and RPCI11.C) are designed. The proximal probe maybe labeled with Spectrum Orange dUTP and the distal probe may be labeledwith Spectrum Green dUTP, Labeling of the probes by nick translation andinterphase FISH using FFPE tissue sections may be done according to themanufacturer's instructions (Vysis Inc., Downers Grove, Ill.) with thefollowing modifications. In brief, paraffin embedded tissue sections arere-hydrated and are subjected to pretreatment first in 0.2N HCl for 20minutes followed by 1 M sodium thiocyanate at 80 C for 30 min.

Following a brief wash, sections are digested with protease (8 mgPepsin, 2000-3000 U/mg) for 45-60 minutes at 37° C. then fixed in 10%NBF and dehydrated. The probe set is then loaded onto the sections andincubated at 94 C for 3 min in order to denature the probe and targetchromosome. Following denaturation the slides are incubated at 37 C fora minimum of 18 hours. After washing, 4′,6-diamidino-2-phenylindole(DAPI; mg/ml) in Vectashield mounting medium (Vector Laboratories,Burlingame, Calif.) will be applied for nuclear counterstaining.

The FIG-ROS rearrangement probe will contain three differently labeledprobes. Two of these probes (RP11-323017, RP1-94G16) target the deletionarea between the break points of the FIG gene and the ROS gene and theother probe (RP1-179P9) targets the remaining portion of the ROS gene(see FIG. 16). The sequences of the introns containing the break pointsof the FIG and ROS genes are provided in SEQ ID NO: 5 (intron 3 of FIG),SEQ ID NO: 6 (intron 7 of FIG), SEQ ID NO: 7 (intron 33 of ROS), SEQ IDNO: 8 (intron 34 of ROS), and SEQ ID NO:26 (intron 31 of ROS). Theprobes are designed based on the breakpoints identified in Example 2.When hybridized, the native (i.e., wild-type) ROS region will appear asan orange/green fusion signal (which may appear yellow under amicroscope), while rearrangement at this locus (as occurs in the FIG-ROSfusion protein) will result in only orange signals since the targetareas for the green probes have been deleted.

For rearrangements of the ROS gene with either CD74 (on chromosome 5) orSLC34A2 (on chromosome 4), because these genes lie on chromosomes otherthan chromosome 6, the native (i.e., wild-type or non-rearranged) ROSregion will appear as an orange/green fusion signal (which may appearyellow under a microscope), while rearrangement at this locus (as occursin the SLC34A2-ROS fusion proteins and the CD74-ROS fusion proteins)will result in a separate orange signal (on chromosome 6) and separategreen signal (on chromosome 5 for CD74 and chromosome 4 for SLC34A2).

The FISH analysis will likely reveal a low incidence of ROS genetranslocations in the sample population having liver cancer (e.g., in acholangiocarcinoma), pancreatic cancer, kidney cancer, or testicularcancer. However, it is predicted that a subset of the studied cancerswill contain a ROS translocation. These cancers containing the FIG-ROStranslocation are identified as those cancers likely to respond to a ROSinhibitor. In other words, cells of the cancer, upon treatment (orcontact) with a ROS inhibitor are predicted to show growth retardation,growth abrogation (i.e., stop growing) or actually die (e.g., byapoptosis) as compared to untreated cancer cells (i.e., cells notcontacted with the ROS inhibitor).

Example 11 Identification of Mutant ROS Expression in Human LiverCancers

Next, studies were performed to determine if ROS expression could beobserved in samples from human liver cancers. The two most common typesof liver cancer are hepatocellular carcinoma (HCC), accounting for 80%of all cases, and cholangiocarcinoma (CCA, or bile duct cancer),representing 10-15% of hepatobiliary neoplasms (Blechacz et al.,Hepatology 48:308-321, 2008 and de Groen, P. C., N Engl J Med341:1368-1378, 1999). For these studies, an ROS-specific antibody (cloneno. D4D6) that specifically bound to the c-terminus of ROS was used.Such antibodies are commercially available (see, e.g., the Ros (C-20)antibody, Catalog No. sc-6347 from Santa Cruz Biotechnology, Inc., SantaCruz, Calif.).

For the studies on cholangiocarcinoma, nineteen human cholangiocarcinomaparaffin-embedded tissue blocks and slides were obtained from BioChainInstitute, Inc., Hayward, Calif., Folio Biosciences, Columbus, Ohio andAnalytical Biological Services, Inc., Wilmington, Del. 4-6 μm tissuesections were deparaffinized through three changes of xylene for 5minutes each, then rehydrated through two changes of 100% ethanol and 2changes of 95% ethanol, each for 5 minutes.

The deparraffinized slides were then rinsed for 5 minutes each in threechanges of diH₂O, then were subjected to antigen retrieval in aDecloaking Chamber (Biocare Medical, Concord, Calif.). Slides wereimmersed in 250 ml 1.0 mM EDTA, pH 8.0 in a 24 slide holder from TissueTek. The Decloaking Chamber was filled with 500 ml diH₂O, the slideholder was placed in the chamber touching the heat shield, and retrievalwas performed with the following settings as set by the manufacturer:SP1 125° C. for 30 seconds and SP2 90° C. for 10 seconds. Slides werecooled on the bench for 10 minutes, rinsed in diH₂O, submerged in 3%H₂O₂ for 10 minutes, then washed twice in diH₂O.

After blocking for 1 hour at room temperature in Tris buffered saline+0.5% Tween-20 (TBST)/5% goat serum in a humidified chamber, slides wereincubated overnight at 4° C. with Ros (D4D6) XP™ Rabbit mAb at 0.19μg/ml diluted in SignalStain® Antibody Diluent (catalog #8112 CellSignaling Technology, Danvers, Mass.). After washing three times inTBST, detection was performed with SignalStain® Boost IHC DetectionReagent (HRP, Rabbit) (catalog #8114 Cell Signaling Technology, Danvers,Mass.) with a 30 minute incubation at room temperature in a humidifiedchamber.

After washing three times in TBST to remove theSignalStain® Boost IHCDetection Reagent, the slides were next exposed to NovaRed (VectorLaboratories, Burlingame, Calif.) prepared per the manufacturer'sinstructions. Slides were developed for 1 minute and then rinsed indiH₂O, Slides were counterstained by incubating in hematoxylin (Ready touse Invitrogen (Carlsbad, Calif.) Catalog #00-8011) for 1 minute, rinsedfor 30 seconds in diH₂O, incubated for 20 seconds in bluing reagent(Richard Allan Scientific, Kalamazoo, Mich. (a Thermo Scientificcompany), Catalog #7301), and then finally washed for 30 seconds indiH₂O, Slides were dehydrated in 2 changes of 95% ethanol for 20 secondseach and 2 changes of 100% ethanol for 2 minutes each. Slides werecleared in 2 changes of xylene for 20 seconds each, then air dried.Coverslips were mounted using VectaMount (Vector Laboratories,Burlingame, Calif.). Slides were air dried, then evaluated under themicroscope.

Of the nineteen samples assayed, six samples stained positive forbinding of the ROS-specific antibody. FIG. 17 shows a representativeimage of slide from a CCA tissue sample that stained positive for ROSexpression. This finding is notable because ROS is not expressed innormal bile duct tissue and is also not expressed in normal livertissue.

Sequencing analysis of the samples showing strong staining with theROS-specific antibody is expected to reveal the presence of eithermutant ROS expression (e.g., over-expression of wild-type ROS in thebile duct cancer tissue where in normal bile duct tissue there is none)or the presence of a truncated ROS polypeptide or a ROS fusion protein(e.g., a FIG-ROS fusion polypeptide).

For studies on hepatocellular carcinoma, 23 paraffin-embedded human HCCtissue array sectioned at 4 μm were deparaffinized through three changesof xylene for 5 minutes each, then rehydrated through two changes of100% ethanol and 2 changes of 95% ethanol, each for 5 minutes. Slideswere rinsed for 5 minutes each in three changes of diH₂O, then weresubjected to antigen retrieval in a Decloaking Chamber (Biocare Medical,Concord, Calif.) as follows. Slides were immersed in 250 ml 1.0 mM EDTA,pH 8.0 in a 24 slide holder from Tissue Tek. The Decloaking Chamber wasfilled with 500 ml diH₂O, the slide holder was placed in the chambertouching the heat shield, and retrieval was performed with the followingsettings as set by the manufacturer: SP1 125° C. for 30 seconds and SP290° C. for 10 seconds. Slides were cooled on the bench for 10 minutes,rinsed in diH₂O, submerged in 3% H₂O₂ for 10 minutes, then washed twicein diH₂O.

After blocking for 1 hour at room temperature in Tris buffered saline+0.5% Tween-20 (TBST)/5% goat serum in a humidified chamber, slides wereincubated overnight at 4° C. with Ros (D4D6) XP™ Rabbit mAb at 0.19μg/ml diluted in SignalStain® Antibody Diluent (#8112 Cell SignalingTechnology, Danvers, Mass.). After washing three times in TBST,detection was performed with SignalStain® Boost IHC Detection Reagent(HRP, Rabbit) (#8114 Cell Signaling Technology, Danvers, Mass.) with a30 minute incubation at room temperature in a humidified chamber.

After washing three times in TBST slides were exposed to NovaRed (VectorLaboratories, Burlingame, Calif.) prepared per the manufacturer'sinstructions. Slides were developed for 1 minute then rinsed in diH₂O,Slides were counterstained by incubating in hematoxylin (Ready to useInvitrogen #00-8011) for 1 minute, rinsed for 30 seconds in diH₂O,incubated for 20 seconds in bluing reagent (Richard Allan Scientific#7301), then finally washed for 30 seconds in diH₂O, Slides weredehydrated in 2 changes of 95% ethanol for 20 seconds

each and 2 changes of 100% ethanol for 2 minutes each. Slides werecleared in 2 changes of xylene for 20 seconds each, then air dried.Coverslips were mounted using VectaMount (Vector Laboratories,Burlingame, Calif.). Slides were air dried, then evaluated under themicroscope.

Of the twenty-three samples assayed, one sample was strongly positivefor staining (i.e., binding) by the ROS-specific antibody and nine casesshowed weak to moderate staining. FIG. 18 shows a representative imageof slide from a HCC tissue sample that stained moderately, positive forROS expression. This finding is notable because ROS is not expressed innormal bile duct tissue and is also not expressed in normal livertissue.

Sequencing analysis of the samples showing strong staining with theROS-specific antibody is expected to reveal the presence of eithermutant ROS expression (e.g., over-expression of wild-type ROS in thehepatocellular carcinoma tissue where there is none in normal livertissue) or the presence of a truncated ROS polypeptide or a ROS fusionprotein (e.g., a FIG-ROS fusion polypeptide).

To determine whether or not the ROS antibody used was able to bindmutant ROS in these liver tissues, an IHC assay was performed on HCC78cells (a non-small cell lung cancer known to express an SLC34A2-ROSfusion polypeptide) in the presence or absence of a competing ROSpeptide.

IHC was performed as described above for the HCC and CCA tissue samples.Briefly, paraffin embedded HCC78 cell pellets were deparaffinized andrehydrated through three changes of xylene and graded ethanol, thenrinsed in diH₂O. Slides were subjected to antigen retrieval in 1.0 mMEDTA, pH 8.0 in the microwave. After blocking for 1 hour in TBST/5% goatserum, slides were incubated overnight at 4° C. with Ros (D4D6) XP™Rabbit mAb at 0.19 μg/ml in the absence of peptide or in the presence ofone of 13 different ROS peptides at 1.9 ug/ml. The ROS peptides were asfollows:

Peptide number: M09-6291Peptide name: ROS-1Peptide sequence: (biotin)AGAGCGQGEEKSEG (SEQ ID NO: 45)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6300Peptide name: ROS-10Peptide sequence: (biotin)AGAGSGKPEGLNYA (SEQ ID NO: 46)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6301Peptide name: ROS-11Peptide sequence: (biotin)AGAGGLNYACLTHS (SEQ ID NO: 47)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6302Peptide name: ROS-12Peptide sequence: (biotin)AGAGCLTHSGYGDG (SEQ ID NO: 48)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6303Peptide name: ROS-13Peptide sequence: (biotin)AGAGTHSGYGDGSD (SEQ ID NO: 49)Peptide carboxyl-terminus: CONH2Synthesis scale (mop: 5Peptide number: M09-6292Peptide name: ROS-2Peptide sequence: (biotin)AGAGEKSEGPLGSQ (SEQ ID NO: 50)Peptide carboxyl-terminus: CONH2Synthesis scale (1 μmol): 5Peptide number: M09-6293Peptide name: ROS-3Peptide sequence: (biotin)AGAGPLGSQESESC (SEQ ID NO: 51)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6294Peptide name: ROS-4Peptide sequence: (biotin)AGAGESESCGLRKE (SEQ ID NO: 52)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6295Peptide name: ROS-5Peptide sequence: (biotin)AGAGGLRKEEKEPH (SEQ ID NO: 53)Peptide carboxyl-terminus: CONH2Synthesis scale (μmmol): 5Peptide number: M09-6296Peptide name: ROS-6Peptide sequence: (biotin)AGAGEKEPHADKDF (SEQ ID NO: 54)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6297Peptide name: ROS-7Peptide sequence: (biotin)AGAGADKDFCQEKQ (SEQ ID NO: 55)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6298Peptide name: ROS-8Peptide sequence: (biotin)AGAGCQEKQVAYCP (SEQ ID NO: 56)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5Peptide number: M09-6299Peptide name: ROS-9Peptide sequence: (biotin)AGAGVAYCPSGKPE (SEQ ID NO: 57)Peptide carboxyl-terminus: CONH2Synthesis scale (μmol): 5

After washing, detection was performed with SignalStain® Boost IHCDetection Reagent (HRP, Rabbit) #8114 and NovaRed (Vector Laboratories,Burlingame, Calif.).

The results show that only peptide 9 was able to compete the binding ofthe antibody off of the IHC slide. FIG. 19A shows an IHC slide with theaddition of peptide ROS-1 and FIG. 19B shows an IHC slide with theaddition of peptide ROS-9. Thus, the sequence of ROS-9, namelyAGAGVAYCPSGKPE (SEQ ID NO: 58), is within the ROS kinase fragmentspecifically bound to by the antibody used in these studies. Since thissequence appears within the kinase domain of the ROS kinase, thesestudies strongly suggest that the CCA and HCC tissues that stainedpositive for binding with the ROS-specific antibody were expressing thekinase domain of ROS.

While the invention has been described with particular reference to theillustrated embodiments, it will be understood that numerousmodifications thereto will appear to those skilled in the art.Accordingly, the above description and accompanying drawings should betaken as illustrative of the invention and not in a limiting sense.

1. A purified FIG-ROS(S) fusion polypeptide, wherein said polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 4 or SEQ IDNO:
 17. 2. A purified FIG-ROS(S) fusion polynucleotide, wherein saidpolynucleotide comprises the nucleic acid sequence set forth in SEQ IDNO: 3 or SEQ ID NO:
 16. 3. A binding agent that specifically binds to aFIG-ROS fusion polypeptide.
 4. The binding agent of claim 3, wherein thebinding agent specifically binds to a fusion junction between a FIGportion and a ROS portion in said FIG-ROS fusion polypeptide.
 5. Thebinding agent of claim 4, wherein the fusion junction comprises an aminoacid sequence selected from the group consisting of AGSTLP, LQVWHR, andLQAGVP.
 6. The binding agent of claim 3, wherein the FIG-ROS fusionpolypeptide is selected from the group consisting of a FIG-ROS(S) fusionpolypeptide, a FIG-ROS(L) fusion polypeptide, and a FIG-ROS(XL) fusionpolypeptide.
 7. The binding agent of claim 3, wherein the binding agentis selected from the group consisting of an antibody and an AQUApeptide.
 8. A nucleotide probe for detecting a FIG-ROS(S)fusionpolynucleotide or a FIG-ROS(XL) fusion polynucleotide, wherein saidprobe hybridizes to said FIG-ROS(S) fusion polynucleotide or to aFIG-ROS(XL) fusion polynucleotide under stringent conditions.
 9. Amethod for detecting a FIG-ROS gene translocation in a biologicalsample, said method comprising contacting a biological sample with abinding agent that specifically binds to a FIG-ROS fusion polypeptide,wherein specific binding of said binding agent to said biological sampleindicates a FIG-ROS gene translocation in said biological sample.
 10. Amethod for detecting a FIG-ROS gene translocation in a biologicalsample, said method comprising contacting a biological sample with anucleotide probe that hybridizes to a FIG-ROS fusion polynucleotideunder stringent conditions, wherein hybridization of said nucleotideprobe to said biological sample indicates a FIG-ROS gene translocationin said biological sample.
 11. A method for diagnosing a patient ashaving a cancer or a suspected cancer characterized by a ROS kinase,wherein said cancer or suspected cancer is not a cancer or suspectedcancer selected from the group consisting of non-small cell lungcarcinoma and glioblastoma, said method comprising contacting abiological sample of said cancer or suspected cancer, said biologicalsample comprising at least one polypeptide, with a binding agent thatspecifically binds to a mutant ROS polypeptide, wherein specific bindingof said binding agent to at least one polypeptide in said biologicalsample identifies said patient as having a cancer or a suspected cancercharacterized by a ROS kinase.
 12. A method for identifying a cancer ora suspected cancer that is likely to respond to a ROS inhibitor, whereinsaid cancer or suspected cancer is not a cancer or suspected cancerselected from the group consisting of non-small cell lung carcinoma andglioblastoma, said method comprising contacting a biological sample ofsaid cancer or suspected cancer, said biological sample comprising atleast one polypeptide with a binding agent that specifically binds to amutant ROS polypeptide, wherein specific binding of said binding agentto at least one polypeptide in said biological sample identifies saidcancer or suspected cancer as a cancer or suspected cancer that islikely to respond to a ROS inhibitor.
 13. The method of claim 11 or 12,wherein said mutant ROS polypeptide is aberrantly expressed wild-typeROS polypeptide.
 14. The method of claim 11 or 12, wherein said mutantROS polypeptide is selected from the group consisting of a truncated ROSpolypeptide and a ROS fusion polypeptide.
 15. The method of claim 14,wherein the ROS fusion polypeptide is selected from the group consistingof a FIG-ROS(S) fusion polypeptide, a FIG-ROS(L) fusion polypeptide, aFIG-ROS(XL) fusion polypeptide, a SLC34A2-ROS(S) fusion polypeptide, aSLC34A2-ROS(L) fusion polypeptide, a SLC34A2-ROS(VS) fusion polypeptide,and a CD74-ROS fusion polypeptide.
 16. The method of claim 11 or 12,wherein the binding agent is selected the group consisting of anantibody or an AQUA peptide.
 17. A method for diagnosing a patient ashaving a cancer or a suspected cancer characterized by a ROS kinase,wherein said cancer or suspected cancer is not a cancer or suspectedcancer selected from the group consisting of non-small cell lungcarcinoma and glioblastoma, said method comprising contacting abiological sample of said cancer or a suspected cancer, said biologicalsample comprising at least one nucleic acid molecule, with a probe thathybridizes under stringent conditions to a nucleic acid moleculeselected from the group consisting of a FIG-ROS fusion polynucleotide, aSLC34A2-ROS fusion polypeptide, a CD74-ROS fusion polypeptide, and atruncated ROS polynucleotide, and wherein hybridization of said probe toat least one nucleic acid molecule in said biological sample identifiessaid patient as having a cancer or a suspected cancer characterized by aROS kinase.
 18. A method for identifying a cancer or a suspected cancerthat is likely to respond to a ROS inhibitor, wherein said cancer orsuspected cancer is not a cancer or suspected cancer selected from thegroup consisting of non-small cell lung carcinoma and glioblastoma, saidmethod comprising contacting a biological sample of said cancer orsuspected cancer, said biological sample comprising at least one nucleicacid molecule, with a probe that hybridizes under stringent conditionsto a nucleic acid molecule selected from the group consisting of aFIG-ROS fusion polynucleotide, a SLC34A2-ROS fusion polypeptide, aCD74-ROS fusion polypeptide, and a truncated ROS polynucleotide, whereinhybridization of said probe to at least one nucleic acid molecule insaid biological sample identifies said cancer or suspected cancer as acancer or suspected cancer that is likely to respond to a ROS inhibitor.19. The method of claim 17 or 18, wherein the FIG-ROS fusionpolynucleotide encodes a fusion polypeptide selected from the groupconsisting of FIG-ROS(S) fusion polypeptide, a FIG-ROS(L) fusionpolypeptide, and a FIG-ROS(XL) fusion polypeptide.
 20. The method ofclaim 17 or 18, wherein the SCL34A2-ROS fusion polynucleotide encodes afusion polypeptide selected from the group consisting of SCL34A2-ROS(S)fusion polypeptide, a SCL34A2-ROS(L) fusion polypeptide, and aSCL34A2-ROS(VS) fusion polypeptide.
 21. The method of claim 11, 12, 17,or 18, wherein said cancer or suspected cancer is selected from thegroup consisting of a kidney cancer, a liver cancer, a pancreaticcancer, and a testicular cancer,
 22. The method of claim 11, 12, 17, or18, wherein the cancer is from a human.
 23. The method of claim 12 or18, wherein the ROS inhibitor is selected from the group consisting of abinding agent that specifically binds to a FIG-ROS fusion polypeptide, abinding agent that specifically binds to a truncated ROS polypeptide, ansiRNA targeting a FIG-ROS fusion polynucleotide, and an siRNA targetinga truncated ROS polynucleotide.
 24. The method of claim 12 or 18,wherein the ROS inhibitor is an inhibitor of a kinase selected from thegroup consisting of an ALK kinase, a LTK kinase, an Insulin Receptor,and an IGF1 Receptor.